Phosphorodiamidates and other phosphorus derivatives of fingolimod and related S1P receptor modulators

ABSTRACT

Compounds of general formula (I): (Formula I)) wherein R1, Q, R3, R4, R5, R6, R7 and Ar1 are as defined herein are inhibitors of class I histone deacetylases and are of use in the treatment of lysosomal storage disorders, especially Niemann-Pick type C disease, as well as other lysosomal storage disorders, defective autophagy, accumulation of free cholesterol and mycobacterial diseases.

TECHNICAL FIELD

The present invention is directed to novel compounds that are useful inthe treatment of Niemann-Pick diseases, particularly Niemann-Pick type Cdisease as well other lysosomal storage disorders, particularlysphingolipidoses; endocytic transport abnormalities, defectiveautophagy, accumulation of free cholesterol, elevated levels ofglycosphingolipids, and mycobacterial infection, such as BCG and TB. Inparticular, the invention relates to phosphoramidates,phosphorodiamidates and other phosphorus derivatives of fingolimod andrelated S1P receptor modulators. The invention also relates to methodsfor preparing the compounds and to pharmaceutical compositionscontaining them.

BACKGROUND

Niemann-Pick diseases are a subgroup of lipid storage disorders calledsphingolipidoses in which harmful quantities of fatty substances, orlipids, accumulate in the spleen, liver, lungs, bone marrow, and brain,and represent a group of severe metabolic disorders in whichsphingomyelin accumulates in lysosomes in cells.

Cholesterol storage and failure in the fusion of lateendosomes/lysosomes (LE/Lys) is a hallmark feature of the Type C form ofthe disease, Niemann-Pick type C (NPC). Niemann-Pick type C affects anestimated 1:150,000 people. Approximately 50% of cases present before 10years of age, but manifestations may first be recognized as late as thesixth decade. Niemann-Pick type C is biochemically, genetically andclinically distinct from Niemann-Pick Types A or and B. In Types A andB, there is complete or partial deficiency of the lysosomal enzymecalled acid sphingomyelinase. In Niemann-Pick type C, the proteinproduct of the major mutated gene NPC1 is not an enzyme but appears tofunction as a transporter in the endosomal-lysosomal system, which moveslarge water-insoluble molecules through the cell.

NPC is caused by mutations in the NPC1 (95% of clinical cases) or NPC2genes, with defects in either gene resulting in identical clinicalphenotypes. NPC1 encodes NPC1, a membrane protein in the LE/Lysmembrane. In contrast, NPC2 is a soluble cholesterol-binding protein ofthe lysosomal lumen. The clinical manifestations of Niemann-Pick typesC1 and C2 are similar because the respective genes are both involved inegress of lipids, particularly cholesterol, from late endosomes orlysosomes. Affected individuals may have enlargement of the spleen(splenomegaly) and liver (hepatomegaly), or enlarged spleen or livercombined (hepatosplenomegaly). Progressive neurological disease is thehallmark of NPC, and is responsible for disability and premature deathin all cases beyond early childhood. A variety of neurological signs andsymptoms typically associate with the disease including cerebellarataxia (unsteady walking with uncoordinated limb movements), dysarthria(slurred speech), dysphagia (difficulty in swallowing), psychosis,progressive dementia, progressive hearing loss, bipolar disorder, majorand psychotic depression that can include hallucinations, delusions,mutism, or stupor. In the terminal stages of NPC, the patient isbedridden, with complete ophthalmoplegia, loss of volitional movementand severe dementia.

Upon the pharmacological inactivation of NPC1 the first measurable eventis an increase in sphingosine levels in the LE/Lys, rapidly followed bydecreased lysosomal Ca²⁺ levels and subsequent attenuated Ca²⁺ releasefrom the LE/Lys. This leads to downstream endocytic trafficking defects,failure in LE/Lys fusion and the subsequent accumulation of cholesteroland glycosphingolipids (GSLs) in a distended endo-lysosomal compartment.In addition to storage of multiple lipids, NPC cells also accumulateautophagic vacuoles, due to a failure in their clearance. Accumulationof cholesterol in lysosomes leads to relative deficiency of thismolecule in multiple membranes and for use in steroid synthesis

There is no known cure for NPC, nor is there any FDA-standard approveddisease modifying treatment.

The NPC1 protein, a putative cholesterol and sphingosine transporter, isdisrupted in Niemann-Pick C1 but it appears that disruption of NPC1 isalso a factor in Niemann-Pick type C2 as well as Niemann-Pick types Aand B and other lysosomal storage disorders such as neuronal ceroidlipofuscinoses (NCL), lipidoses, mucolipidoses and sphingolipidoses suchas, Gaucher disease, Fabry disease and Tay-Sachs disease. It has alsobeen shown that the NPC1 protein is altered in other diseases leading tothe accumulation of free cholesterol, these include conditions such asSmith-Lemli-Opitz syndrome and Huntington's where cholesterol precursorsinhibit NPC1 function and in mycobacterial diseases such as tuberculosisand Bacillus Calmette-Guérin (BCG) where cell wall components of thebacteria mimic cholesterol and inhibit NPC1 function.

Fingolimod (Gilenya; FTY720) is a synthetic compound derived from animmunosuppressive natural fungal secondary metabolite, myriocin (ISP-I),and was first synthesised in 1992 by Yoshitomi Pharmaceuticals and hasthe structure:

Fingolimod was the first drug to be approved for oral administration forthe treatment of relapsing-remitting forms of multiple sclerosis. Inparticular, it has been found to reduce relapses and delay disabilityprogression. More recently, fingolimod has been investigated to treat awide variety of other conditions, including cancer, and has also beenfound to have potential in reducing the incidence of transplantrejection.

Fingolimod becomes active in vivo following phosphorylation bysphingosine kinase 2 to form fingolimod-phosphate (FTY720-P), whichbinds to extracellular G protein-coupled receptors, S1P₁, S1P₃, S1P₄ andS1P₅ receptors, and prevents the release of lymphocytes from lymphoidtissue.

Recently it has been shown that FTY720 enters the nucleus, where it isphosphorylated by sphingosine kinase 2 (SphK2), and then nuclearFTY720-P binds and inhibits class I histone deacetylases (HDACs),enhancing specific histone acetylations. FTY720 is also phosphorylatedin mice and accumulates in the brain, including the hippocampus, whereit inhibits HDACs and enhances histone acetylation and gene expressionprograms associated with memory and learning, and rescues memorydeficits independently of its immunosuppressive actions. Sphk2−/− micehave lower levels of hippocampal sphingosine-1-phosphate, an endogenousHDAC inhibitor, and reduced histone acetylation, and display deficits inspatial memory and impaired contextual fear extinction.

It has recently been shown that administration of clinically relevantdoses of FTY720 to mice increased expression of NPC1 and −2 in brain andliver and decreased cholesterol in an SphK2-dependent manner (Newton etal, FASEB J. 2017 Apr.; 31(4): 1719-1730). FTY720 greatly increasedexpression of NPC1 and −2 in human NPC1 mutant fibroblasts thatcorrelated with formation of FTY720-P and significantly reduced theaccumulation of cholesterol and glycosphingolipids. However, a problemwith the use of fingolimod is that it must be phosphorylated in the cellby sphingosine kinases before it can bind to the S1P receptors. Thisrequirement may limit its efficacy or impose tissue selectivity ofaction, as not all individuals possess the relevant kinase necessary foractivation thus rendering treatment in such individuals ineffective.More substantially, sphingoid bases such as sphingosine, a family towhich fingolimod belongs, are known to disrupt NPC1 function(particularly at higher concentrations) and induce lysosomal expansionpotentially therefore reversing the beneficial effects of fingolimod(Lloyd-Evans et al, Nature Medicine, 14, 1247-1255, 2008). Thispotentially adverse effect is also more likely because of anaccumulation of fingolimod in the cells during the course of treatmentleading thus to increased inhibition of NPC1 and reduced efficacy of thedrug.

The inventors have designed novel derivatives of fingolimod and variousanalogues thereof, which are capable of acting as prodrugs for the knownbio-active phosphate forms of the compound. Advantageously, thesederivatives and analogues, or compounds, have been found to haveincreased uptake into target cells whilst avoiding the negative effectsattributed to accumulation and lysosomal expansion as per the parentcompound. Consequently, these compounds potentially represent superiortherapeutics for use in the treatment of lysosomal storage disorderssuch as mucolipidoses, lipidoses, including NPC and Niemann-Pick type C2(NPC2); neuronal ceroid lipofuscinoses (NCL), for example NCL types1-10; and sphingolipidoses such as, Niemann-Pick types A and B, Gaucherdisease, Fabry disease and Tay-Sachs disease; more especially NPC andparticularly NPC1.

Notably, many NPC cellular phenotypes are also observed includingendocytic transport abnormalities, defective autophagy, accumulation offree cholesterol, elevated levels of glycosphingolipids, andmycobacterial infection, such as BCG, and TB. We have found thatinfection with persistent intracellular mycobacteria, such as BCG andTB, induces the full range of NPC phenotypes in wild-type cells, andlipids shed by these mycobacteria were able to phenocopy NPC diseasecellular phenotypes in the absence of the mycobacteria itself.Furthermore, therapies developed for the treatment of NPC diseasepromoted mycobacterial clearance, suggesting the compounds of theinvention could also be used to treat any of the afore diseases orconditions, in particular mycobacterial infection, such as TB. We havealso found that in Huntington's disease, the mutant Htt protein isnecessary to transport NPC1 to lysosomes, leading to the presence of allNPC disease phenotypes and the potential for NPC therapies to work forHD.

SUMMARY

Therefore, in a first aspect of the present invention there is provideda compound of general formula (I) including all stereoisomers thereofand all isotopic variants thereof:

wherein

R¹ is —OAr² or -Q′R³′;

-   -   wherein Ar² is a C₆₋₁₀ aryl or a 5-10 membered heteroaryl group        optionally substituted with one or more substituents selected        from OH, halo, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —O(C₁₋₆        alkyl), —O(C₁₋₆ haloalkyl), NH₂, NH(C₁₋₆ alkyl), N(C₁₋₆ alkyl)₂        or SF₅;

Q and Q′ are each independently O, S or NR²;

R² is H or C₁₋₆ alkyl optionally substituted by one or more halo, OH orphenyl substituents;

R³ and R³′ are each independently C₁₋₁₀ alkyl or C₁₋₁₀ alkyl—C(O)OR¹¹,either or which is optionally substituted by one or more substituentsR¹²,

-   -   R¹¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl or benzyl;    -   R¹² is —O—R¹³, —SR¹³, Z, —Z—O—R¹³, —O—Z—R¹³, —Z—R¹³, —C(O)R¹³,        —C(O)OR¹³, NR¹³R¹⁴, C(O)NR¹³R¹⁴, —NHC(O)R¹³, —NHC(O)OR¹³,        NH(C═NH)NR¹³R¹⁴, —OC(O)—R¹³, —SC(O)R¹³ or —S—S—R¹³;        -   R¹³ and R¹⁴ are each independently H or C₁₋₆ alkyl;        -   Z is a C₆₋₁₀ aryl or a 5- to 10-membered heteroaryl group            optionally substituted with one or more substituent selected            from halo or OH;

or when Q or Q′ is NR², R² and R³ or R² and R³′ together with thenitrogen atom to which they are attached, form a 5- or 6-memberedheterocyclic ring substituted with C(O)OR¹¹, wherein R¹¹ is as definedabove;

R⁴ is OH or a group:

where R¹, Q and R³ are as defined above;

each of R⁵ and R⁶ is independently selected from hydrogen or C₁₋₄ alkyl;or R⁵ and R⁶ together with the nitrogen atom to which they are attachedmay form a 5- or 6-membered heterocyclic ring optionally containing afurther heteroatom selected from N, O or S;

Ar¹ is a phenyl or a 5- or 6-membered heteroaryl group, either of whichis optionally substituted with one or more substituents selected fromhalo, OH, C₁₋₄ alkyl or C₁₋₄ haloalkyl; and

R⁷ is C₁₋₁₀ alkyl optionally substituted with phenyl or a 5- or6-membered heteroaryl group, wherein the phenyl or heteroaryl groups areoptionally substituted with one or more substituents selected from halo,NO₂, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, O(C₁₋₄ alkyl) or phenyl optionallysubstituted with halo, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl or O(C₁₋₄ alkyl),and is optionally labelled with a detectable label;

or a pharmaceutically or veterinarily acceptable salt or hydratethereof.

The compounds of the present invention affect the NPC1 pathway, suchthat they are suitable for treating lysosomal storage disorders as wellas defective autophagy, accumulation of free cholesterol, elevatedlevels of glycosphingolipids, and mycobacterial infection. In addition,there is evidence to suggest that they may also affect neuronal ceroidlipofuscinoses. The compounds of the present invention appear to besuperior to fingolimod, the parent compound from which they are derived.Without wishing to be bound by theory, the inventors postulate that thisis because the compounds of the invention do not require phosphorylationby sphingosine kinases. Since the phosphorylation appears to be alimiting step, this means that more of the pharmacophore is madeavailable for binding to the S1P receptors with the compounds of theinvention than with the parent compound.

In the present specification, the term “C₁-C₆ alkyl” refers to astraight or branched saturated hydrocarbon group having one to sixcarbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl,t-butyl, n-hexyl,

The terms “C₁₋₄ alkyl” and “C₁₋₁₀ alkyl have similar meanings exceptthat they have 1-4 or 1-10 carbon atoms respectively and alkyl groupswith other numbers of carbon atoms can be identified using suchnotation.

In the present specification “C₁₋₆ haloalkyl” refers to a C₁₋₆ alkylgroup substituted with one or more halo atoms, up to per-substitution.Examples include chloromethyl, trifluoromethyl, 1,2-dibromoethyl.

The term “C₆₋₁₀ aryl” in the context of the present specification referto a ring system with aromatic character having from 6 to 10 ring carbonatoms and containing a single ring or two fused rings. Where an arylgroup contains two fused rings, both rings need not be fully aromatic incharacter. Examples of aromatic moieties are phenyl, naphthalene,tetrahydronaphthalene, indane and indene. C₆₋₁₄ aryl groups are asdefined above but have from 6 to 14 ring carbon atoms. Examples includeanthracene and fluorene.

The term “heteroaryl” in the context of the specification refer to aring system with aromatic character having from 5 to 14 ring atoms(unless otherwise specified), at least one of which is a heteroatomselected from N, O and S, and containing up to three rings. Where aheteroaryl group contains more than one ring, not all rings must befully aromatic in character. Examples of heteroaryl groups includepyridine, pyrimidine, indole, pyrrole, imidazole, triazole, tetrazole,oxazole, thiazole, benzofuran, benzimidazole and indolene.

The terms “heterocyclic” and “heterocyclyl” in the context of thespecification refer to a ring system having 5-7 ring atoms (unlessotherwise specified), at least one of which is a heteroatom selectedfrom N, O and S, and which is not aromatic in character. Examplesinclude piperidine, piperazine, pyrrolidine, morpholine andtetrahydrofuran.

In the present specification “halo” refers to fluoro, chloro, bromo oriodo.

Appropriate pharmaceutically and veterinarily acceptable salts of thecompounds of general formulae (I) include basic addition salts such assodium, potassium, calcium, aluminium, zinc, magnesium and other metalsalts as well as choline, diethanolamine, ethanolamine, ethyl diamine,megulmine and other well known basic addition salts as summarised inPaulekuhn et al., (2007) J. Med. Chem. 50: 6665-6672 and/or known tothose skilled in the art.

Salts which are not pharmaceutically or veterinarily acceptable maystill be valuable as intermediates.

Suitably, in the compounds of general formula (I), the asymmetric carbonatom (*) to which the NR⁵R⁶, CH₂R⁴, —CH₂CH₂—Ar¹—R⁷ and the phosphatemoiety:

are attached is in the S-orientation.

In some suitable compounds of the present invention, R¹ is OAr².Suitably, in such compounds, Ar² is an aryl group such as phenyl,naphthyl, for example 1-naphthyl, or tetrahydronaphthyl, for example5,6,7,8-tetahydro-1-naphthyl, any of which is optionally substitutedwith one or more substituents as set out above. When Ar² is asubstituted phenyl group, the substituents are suitably in the 2- and/or4- and/or 6-positions, particularly the 4-position.

In some compounds, the group Ar² is unsubstituted.

In other suitable compounds, R¹ is -Q′R³′.

In some suitable compounds Q and/or Q′ (when present) is O.

In some suitable compounds Q and/or Q′ (when present) is S.

In some suitable compounds Q and/or Q′ (when present) is NR² where R² isH or C₁₋₄ alkyl optionally substituted with one or more halo, OH orphenyl substituents. In some cases, R² is H or unsubstituted C₁₋₄ alkyl,for example H, methyl or ethyl but especially H or methyl.

In the compounds of the invention, R³ and/or R³′ (when present) issuitably a group C₁₋₁₀ alkyl—C(O)OR¹¹. Such R³ and R³′ groups areparticularly suitable in the case where Q or Q′ is NR².

In some more suitable compounds, R³ and/or R³′ (when present) may be—C(R^(12a)R^(12b))C(O)OR¹¹ or —C(R^(12a)R^(12b))CH₂C(O)OR¹¹;

wherein R¹¹ is as defined above;

R^(12a) is H or C₁₋₆ alkyl optionally substituted by a group R¹² asdefined above;

R^(12b) is H, methyl or ethyl, more suitably H or methyl and especiallyH.

In some cases, R³ and/or R³′ (when present) is—C(R^(12a)R^(12b))CH₂C(O)OR¹¹, where both R^(12a) and R^(12b) are H,such that R³ and/or R³′ (when present) is —CH₂CH₂C(O)OR¹¹.

Compounds in which R³ and/or R^(3′) (when present) is—C(R^(12a)R^(12b))C(O)OR¹¹ are particularly suitable.

Often, R^(12a) is an amino acid side chain. In some cases, R^(12a) maybe a side chain of a naturally-occurring amino acid, where the naturallyoccurring amino acid may be alanine, valine, leucine, isoleucine,methionine, phenylalanine, tyrosine, tryptophan, arginine, histidine,lysine, aspartic acid, glutamic acid, serine, threonine, asparagine,glutamine, cysteine, glycine or proline.

Usually, when R^(12a) is an amino acid side chain, it hasL-stereochemistry.

Alternatively, R^(12a) may be a side chain of an amino acid havingD-stereochemistry, for example D-alanine.

R^(12a) may suitably be a side chain of alanine, glycine, valine,leucine, isoleucine, phenylalanine, tyrosine, methionine or tryptophan,for example a side chain of alanine such that R^(12a) is methyl.

Suitably R^(12b) is C₁₋₄ alkyl or H, especially H.

In some cases, the amino acid side chains may be modified such that OHand/or SH groups are replaced with O—C₁₋₆ alkyl or S—C₁₋₆ alkyl and/orcarboxylic acid groups are esterified as a C₁₋₆ alkyl or benzyl ester.

Therefore, suitably, in the compounds where R³ and/or R³′ (when present)is substituted with one or more substituents R¹², when present, each ofR¹³ and R¹⁴ is C₁₋₆ alkyl, more usually C₁₋₄ alkyl, for example methylor ethyl and especially methyl.

When Q is NR², R² and R³ together with the nitrogen atom to which theyare attached, form a 5- or 6-membered heterocyclic ring substituted withC(O)OR¹¹, wherein R¹¹ is as defined above.

Similarly when R¹ is Q′R^(3′) and Q′ is NR², R² and R³′ together withthe nitrogen atom to which they are attached, form a 5- or 6-memberedheterocyclic ring substituted with C(O)OR¹¹, wherein R¹¹ is as definedabove.

Suitably, the ring is a 5-membered ring, for example a pyrrolidinylring.

More suitably, the ring is a pyrrolidin-1-yl ring substituted at the2-position with C(O)OR¹¹, wherein R¹¹ is as defined above. Compounds ofthis type are proline derivatives.

In some other suitable compounds of the present invention, R³ is C₁₋₁₀alkyl substituted with, —OC(O)—R¹³, —SC(O)R¹³ or —S—S—R¹³, where R¹³ isH or C₁₋₆ alkyl, more usually C₁₋₆ alkyl. These R³ groups are typicallyfound in compounds of general formula (I) in which Q is O or S,particularly O.

More suitably R³ is C₁₋₆ alkyl substituted with —OC(O)—R¹³, —SC(O)R¹³ or—S—S—R¹³, for example C₁₋₄ alkyl substituted with —OC(O)—R¹³, —SC(O)R¹³or —S—S—R¹³. Examples of this type of R³ group include—CH₂CH₂—OC(O)—(C₁₋₆ alkyl), —CH₂CH₂—SC(O)—(C₁₋₆ alkyl) and—CH₂CH₂—S—S—(C₁₋₆ alkyl).

In some suitable compounds of the present invention at least one of R⁵and R⁶ is H. More suitably, both R⁵ and R⁶ are H.

Suitable Ar¹ groups include phenyl which may be substituted with one ormore halo, methyl, ethyl, halomethyl or haloethyl substituents.Particularly suitable substituents include fluoro, chloro, methyl, ethyland trifluoromethyl.

When Ar¹ is phenyl, the —R⁷ moiety is suitably positioned at the 2-, 4-or 6-position of the phenyl ring with respect to the —CH₂CH₂— linkergroup. Most suitably, the —R⁷ moiety is positioned at the 4-position ofthe phenyl ring with respect to the —CH₂CH₂— linker group.

Other suitable Ar¹ groups include 5- or 6-membered heteroaryl groupssuch as pyridine, pyrimidine, or pyrrole, any of which may besubstituted as described above, particularly with one or more halo,methyl, ethyl, halomethyl or haloethyl substituents.

One example of this type of Ar¹ group is pyrrole, which is optionallysubstituted as described above. In some cases, a pyrrole group may beN-substituted with a C₁₋₄ alkyl group, for example a methyl or ethylgroup and especially with a methyl group.

In some compounds of the present invention, R⁷ is C₁₋₁₀ alkyl,especially C₆₋₁₀ alkyl. Suitably, the alkyl group is a straight chainalkyl group and particularly suitable examples of such groups includen-heptyl or n-octyl groups. R⁷ is most suitably n-octyl.

In other compounds, R⁷ is C₁₋₁₀ alkyl optionally substituted with phenylor a 5- or 6-membered heteroaryl group, wherein the phenyl or heteroarylgroup is optionally substituted with one or more substituents selectedfrom optionally substituted with one or more substituents selected fromhalo, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, O(C₁₋₄ alkyl) or phenyl optionallysubstituted with halo, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl or O(C₁₋₄ alkyl).

More suitably in such compounds, R⁷ is C₁₋₆ alkyl, especially C₁₋₄alkyl, optionally substituted with phenyl or a 5- or 6-memberedheteroaryl group, wherein the phenyl or heteroaryl group is optionallysubstituted as described above. More suitable substituents include C₁₋₄alkyl or phenyl substituted with C₁₋₄ alkyl. In particularly suitablecompounds, the phenyl or heteroaryl group is unsubstituted or issubstituted with methyl, ethyl or phenyl which is unsubstituted orsubstituted with a methyl or ethyl group.

In this type of compound, R⁷ is more suitably an alkyl group substitutedwith a phenyl group, wherein the phenyl group is optionally substitutedas described above. Particularly suitable R⁷ groups include C₁₋₆ alkylsubstituted with phenyl, tolyl or biphenyl.

In some cases, R⁷ is labelled with a detectable label. In other cases R⁷is not labelled with a detectable label.

Detectable labels are well known in the art and include fluorescentlabels, visible labels or isotopic labels.

Fluorescent labels are well known and include derivatives of aromatic orheteroaromatic compounds such as xanthene, naphthaline, coumarin,anthracene, benzo[c][1,2,5]oxadiazole, pyrene or acridine. Other knownfluorescent labelling compounds are based on molecules such as cyanine.

Visible labels are also known and include dyes and coloured beads.

Fluorescent and visible labels may be covalently attached to the R⁷moiety, for example via an amide bond.

The R⁷ moiety may also be isotopically labelled such that one or moreatoms in the R⁷ moiety is replaced by an atom which is chemically thesame but which has a different molecular weight. For example, one ormore of the carbon atoms may be ¹⁴C or one or more hydrogen atoms may be²H or ³H.

Fluorescent labels are particularly suitable.

In suitable compounds of the present invention, the moiety:

is a residue of an S1P modulator of formula:

Examples of such compounds include fingolimod, which has the followingstructure:

Particularly suitable compounds of general formula (I) are compounds inwhich, independently or in any combination:

R⁴ is OH;

R⁵ is H;

R⁶ is H;

R¹ is —OAr¹ and Ar¹ is phenyl;

the R⁷ moiety is positioned at the 4-position of the phenyl ring withrespect to the —CH₂CH₂— linker group;

R⁷ is C₆₋₁₀ alkyl or C₃₋₅ alkyl substituted with (C₁₋₂ alkyl) phenyl;

the C* centre has S stereochemistry.

Especially suitable are compounds in which independently or in anycombination:

R⁴ is OH

R⁵ is H;

R⁶ is H;

R′ is —OAr¹ and Ar¹ is phenyl;

the R⁷ moiety is positioned at the 4-position of the phenyl ring withrespect to the —CH₂CH₂— linker group;

R⁷ is n-octyl; and

the C* centre has S stereochemistry.

Some particularly suitable compounds of general formula (I) arederivatives of Fingolimod, in which the moiety—O—CH₂—C(CH₂OH)(NR⁵R⁶)—CH₂CH₂—Ar¹—R⁷ is

and especially the S enantiomer:

Examples of compounds of general formula (I) include:

(2S)-methyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)propanoate;benzyl 2-(((2-amino-2(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate;

(2S) benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate;

(2S) benzyl-1-((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)pyrrolidine-2-carboxylate;

(2S)ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-propanoate;

(2R)-benzyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;

(2R)-neopentyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino) propanoate; methyl3-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;

(2R)-neopentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)phosphoryl) amino)propanoate;isopropyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate;

(2R)-benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;

(2S)-ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-(methylthio) butanoate;

(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(4-(tert-butoxy)phenyl)propanoate;

(2R)-dimethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)pentanedioate;

(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(1H-indol-3-yl)propanoate;

(2S,3R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)-3-(tert-butoxy)butanoate;

(2R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-6-((tert-butoxycarbonyl) amino)hexanoate;

S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate;

pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate;

methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate;

ethyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate;

(3S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-methylpentanoate;

(2S) pentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate;

and their pharmaceutically acceptable salts, esters and hydrates and allstereochemistries.

The compounds of general formula (I) may be prepared by the methodsdescribed below. As will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and de-protecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts, Protecting Groups in OrganicSynthesis, Third Edition, Wiley, New York, 1999, and references citedtherein.

Compounds of general formula (I) in which R⁵ and R⁶ are both H may beprepared from analogues of the compounds of general formula (I) in whichone of R⁵ and R⁶ is replaced with —C(O)OR¹⁵, wherein R¹⁵ is C₁₋₆ alkylor C₆₋₁₄ aryl optionally substituted with one or more substituentsselected from C₁₋₆ alkyl, C₁₋₆ haloalkyl or halo;

by removal of the protecting group, for example by hydrogenation using asuitable catalyst, such as palladium/carbon. The reaction may beconducted in a solvent such as methanol. The preparation of theseanalogues is described below.

Compounds of general formula (I) may be prepared by the reaction of acompound of general formula (II):

wherein R⁵, R⁶, Ar¹ and R⁷ are as defined for general formula (I);

with a compound of general formula (III):

wherein:

Q, R¹ and R³ are as defined for general formula (I); and

X¹ is halo, particularly chloro.

When Q is NR², O and S the compound of general formula (III) will be acompound of general formula (IIIa), (IIIb) or (IIIc) respectively:

The compound of general formula (II) may firstly be reacted with ahindered base, for example a Grignard reagent, following which theproduct is reacted with the compound of general formula (III). Thereaction may be conducted in an anhydrous organic solvent, for exampletetrahydrofuran and at a temperature of about 15 to 25° C., typically atroom temperature.

This reaction give rise to a mixture of mono compounds of generalformula (I) in which R⁴ is OH and bis compounds of general formula (I)in which R⁴ is a group:

The products can be separated by standard methods, for example HPLC orLC-MS.

This method for the preparation of a compound of general formula (I) isitself an aspect of the invention.

Many compounds of general formula (II) are well known pharmaceuticalagents and are readily available. Other compounds of general formula(II) can readily be synthesised by a person of skill in the art usingstandard methods.

Analogues of compounds of general formula (II) in which one of R⁵ and R⁶is replaced with —C(O)OR¹⁵, wherein R¹⁵ is as defined above, may beprepared from compounds of general formula (II) in which R⁵ and R⁶ areboth H by reaction with a compound of general formula (IX):

wherein R¹⁵ is as defined for general formula (I) and X⁴ is halo,particularly chloro.

Suitably, the reaction is carried out in the presence of a base such assodium bicarbonate and in an organic solvent such as chloroform.

These analogues may be converted into analogues of the compounds ofgeneral formula (I) wherein one of R⁵ and R⁶ is replaced with —C(O)OR¹⁵by reaction with a compound of formula (III) as defined above.

Compounds of general formula (III) may be prepared from a compound ofgeneral formula (IV):

wherein R¹ is as defined for general formula (I), X¹ is as defined forgeneral formula (III) and X² is halo, particularly chloro;

by reaction with a compound of general formula (V):H-Q-R³  (V).

Compounds of general formula (V) in which Q is NR², O and S have,respectively, formulae (Va), (Vb) and (Vc)

wherein R² and R³ are as defined for general formula (I).

Suitably the reaction is carried out in an anhydrous organic solventsuch as dichloromethane and in the presence of a Lewis base such astrimethylamine. The compound of general formula (IV) may be added to asolution of the compound of general formula (V) which is suitably cooledto a temperature of about −78° C., stirred at this temperature for 15 to60 minutes and then allowed to warm to 15-25° C. (room temperature) andthe reaction is continued for 1-4 hours.

Compounds of general formula (V) are well known in the art. For examplecompounds of general formula (Va) may be amino acids, amino acidderivatives or amines. These compounds are readily available or may beprepared by well-known methods. Compounds of general formulae (Vb) and(Vc) are also known or may be synthesised by known methods, for exampleas set out in Example 19 below.

Compounds of general formula (IV) may be prepared from compounds ofgeneral formula (VI):Ar²—OH  (VI)

wherein Ar², R⁸ and R⁹ are as defined for general formula (I);

by reaction with a compound of general formula (VIII):

wherein X¹ is as defined for general formula (III), X² is as defined forgeneral formula (IV) and X³ is halo, particularly chloro. The compoundof general formula (VII) may be used as a suitable salt form.

Suitably the reaction is carried out in an anhydrous organic solventsuch as diethyl ether and in the presence of a Lewis base such astrimethylamine. The reagents are mixed at low temperature, typicallyabout −78° C., stirred at this temperature for 15 to 60 minutes and thenallowed to warm to 15-25° C. (room temperature) and the reaction iscontinued for 1-4 hours.

Compounds of general formulae (VI), (VII) and (VIII) are well known andreadily available. Phosphorus oxychloride is a particularly suitableexample of a compound of general formula (VIII).

As discussed above, the compounds of the present invention are usefulfor the treatment of conditions affected by the NPC1 pathway. Thisincludes lysosomal storage disorders including Niemann-Pick C1,Niemann-Pick type C2, Niemann-Pick types A and B and other lysosomalstorage disorders such as neuronal ceroid lipofuscinoses (NCL),mucolipidoses, lipidoses and sphingolipidoses such as, Gaucher disease,Fabry disease and Tay-Sachs disease; defective autophagy, accumulationof free cholesterol, which leads to conditions such as Smith-Lemli-Opitzsyndrome and mycobacterial diseases such as tuberculosis and BCG as wellas Huntington's disease.

Fingolimod type compounds have been previously tested on NPC cells andappear to have benefits. This is assumed to be due to their role as HDACinhibitors, leading to upregulation of the NPC1 protein. However,fingolimod is similar to sphingosine, which is known to be an earlystorage molecule in NPC, which means that there is potential for it toinitiate downstream lipid storage when used at higher concentrations,thus cancelling out its beneficial effects. The compounds of theinvention are believed to be more effective than the parent compoundsfrom which they are derived because they do not require phosphorylationby sphingosine kinase in order to be converted to the active form. Theinventors have demonstrated that when NPC1-null cells are treated withthe compounds of the invention, the compounds reduced lysosomalexpansion and lipid storage to a greater extent than fingolimod. Itappears that the compounds of the invention may also act on one or moreof the neuronal ceroid lipofuscinoses.

Furthermore, the inventors have demonstrated that the compounds havegood stability in human plasma, with a half-life for some compounds ofaround 7 hours, meaning that the compounds are suitable for once ortwice daily dosing. Furthermore, the inventors have demonstrated thatthe compounds are stable both in acidic and basic conditions, so thatthey are likely to be suitable for use in an oral dosage form.

Therefore, in a further aspect of the invention there is provided acompound of general formula (I) for use in medicine.

There is also provided a compound of general formula (I) for use in thetreatment of lysosomal storage disorders including Niemann-Pick C1,Niemann-Pick type C2, Niemann-Pick types A and B and other lysosomalstorage disorders such as neuronal ceroid lipofuscinoses (NCL),mucolipidoses, lipidoses and sphingolipidoses such as, Gaucher disease,Fabry disease and Tay-Sachs disease; defective autophagy, accumulationof free cholesterol and endocytic transport defects, which leads toconditions such as Smith-Lemli-Opitz syndrome and contributes topathogenesis in Huntington's disease, or mycobacterial diseases such astuberculosis and BCG.

Further, the invention provides the use of a compound of general formula(I) in the preparation of an agent for the treatment of lysosomalstorage disorders including Niemann-Pick C1, Niemann-Pick type C2,Niemann-Pick types A and B and other lysosomal storage disorders such asneuronal ceroid lipofuscinoses (NCL), mucolipidoses, lipidoses andsphingolipidoses such as, Gaucher disease, Fabry disease and Tay-Sachsdisease; defective autophagy, accumulation of free cholesterol andendocytic transport defects, which leads to conditions such asSmith-Lemli-Opitz syndrome and contributes to pathogenesis inHuntington's disease, or mycobacterial diseases such as tuberculosis andBCG.

In another aspect, the invention provides a method for the treatment oflysosomal storage disorders including Niemann-Pick C1, Niemann-Pick typeC2, Niemann-Pick types A and B and other lysosomal storage disorderssuch as neuronal ceroid lipofuscinoses (NCL), mucolipidoses, lipidosesand sphingolipidoses such as, Gaucher disease, Fabry disease andTay-Sachs disease; defective autophagy, accumulation of free cholesteroland endocytic transport defects, which leads to conditions such asSmith-Lemli-Opitz syndrome and contributes to pathogenesis inHuntington's disease, or mycobacterial diseases such as tuberculosis andBCG, the method comprising administering to a patient in need of suchtreatment an effective amount of a compound of general formula (I).

Preferably, the disease to be treated is selected from lysosomal storagedisorders including Niemann-Pick C1, Niemann-Pick type C2, Niemann-Picktypes A and B and other lysosomal storage disorders such as neuronalceroid lipofuscinoses (NCL), lipidoses and sphingolipidoses such as,Gaucher disease, Fabry disease and Tay-Sachs disease; defectiveautophagy, accumulation of free cholesterol and endocytic transportdefects, which leads to conditions such as Smith-Lemli-Opitz syndromeand contributes to pathogenesis in Huntington's disease, ormycobacterial diseases such as tuberculosis and BCG.

In some cases, the disease to be treated is Niemann-Pick disease,particularly NPC and especially NPC1.

In other cases, the disease to be treated is Gaucher disease, Fabrydisease or Tay-Sachs disease

In other cases, the disease to be treated is accumulation of freecholesterol or an endocytic transport defect, for example inSmith-Lemli-Opitz syndrome or Huntington's disease.

In still other cases, the disease to be treated is a mycobacterialinfection, particularly tuberculosis or BCG and especially TB.

The compounds of the present invention will usually be administered in apharmaceutical composition and therefore in a further aspect of theinvention there is provided a pharmaceutical composition comprising acompound of general formula (I) and a pharmaceutically acceptableexcipient or carrier.

The composition may be administered by any appropriate route, forexample oral, buccal, nasal, transdermal or parenteral, for exampleintravenous or intramuscular.

As discussed above, the inventors have shown that many compounds of thepresent invention are stable in acidic environment, with a half-life ofover 12 hours in a solution buffered to pH 1.5 to mimic conditions inthe stomach. The compounds of the invention also have good stability ina basic environment with a half-life of about 13 hours in simulatedintestinal fluid at pH8. In view of this, the compounds are particularlysuited for oral administration.

The compounds of general formula (I) in which R¹ is OAr² are, ingeneral, particularly stable. In the case of such compounds which areunstable, they decompose to form compounds of general formula (I) inwhich R¹ and R⁴ together with the atoms to which they are attached forma 6-membered heterocyclic ring. This compound eventually is hydrolysedto fingolimod monophosphate, the pharmacologically active compound

Formulations for oral administration in the present invention may bepresented as: discrete units such as capsules, sachets, tablets, trochesor lozenges each containing a predetermined amount of the active agent;as a powder or granules; as a solution or a suspension of the activeagent in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water in oil liquid emulsion; or as asyrup or elixir; or as a bolus, etc.

For compositions for oral administration (e.g. tablets, capsules,formulations comprising a mucoadherent etc), the term “acceptablecarrier” includes vehicles such as common excipients e.g. bindingagents, for example syrup, acacia, gelatin, sorbitol, tragacanth,polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sucrose andstarch; fillers and carriers, for example corn starch, gelatin, lactose,sucrose, microcrystalline cellulose, kaolin, mannitol, dicalciumphosphate, sodium chloride and alginic acid; wetting agents/surfactantssuch as poloxamers, polysorbates, sodium docusate and sodium laurylsulfate; disintegrants such as starch or sodium starch glycolate; andlubricants such as magnesium stearate, sodium stearate and othermetallic stearates, glycerol stearate, stearic acid, silicone fluid,talc waxes, oils and colloidal silica. Sweetening agents and flavouringagents such as peppermint, oil of wintergreen, cherry flavouring and thelike can also be used. It may be desirable to add a colouring agent tomake the dosage form readily identifiable. Tablets may also be coated bymethods well known in the art.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active agent in a free flowingform such as a powder or granules, optionally mixed with a binder,lubricant, inert diluent, preservative, surface-active or dispersingagent. Moulded tablets may be made by moulding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active agent.

Some formulations may comprise a mucoadherent, for example amucopolysaccharide such as sodium hyaluronate. Such compositions may beformulated as, for example, liquids, liquid syrups, soft gels, liquidgels, flowable gels or aqueous suspensions and may, in addition to theactive agent and the mucoadherent, also contain one or more additionalexcipients as set out above. Liquid formulations will usually alsocontain a liquid carrier, which may be a solvent or suspending agent,for example water or saline solution and may also contain a substance toincrease their viscosity, for example sodium carboxymethylcellulose,sorbitol or dextran.

Other formulations suitable for oral administration include lozengescomprising the active agent in a flavoured base, usually sucrose andacacia or tragacanth; pastilles comprising the active agent in an inertbase such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active agent in a suitable liquid carrier.

For topical application to the skin, the composition may be made up intoa cream, ointment, jelly, solution or suspension etc. Cream or ointmentformulations that may be used for the drug are conventional formulationswell known in the art, for example, as described in standard text booksof pharmaceutics such as the British Pharmacopoeia.

The composition defined above may be used for the treatment of therespiratory tract by nasal, bronchial or buccal administration of, forexample, aerosols or sprays which can disperse the pharmacologicalactive ingredient in the form of a powder or in the form of drops of asolution or suspension. Pharmaceutical compositions withpowder-dispersing properties include dry powder inhalers and metereddose inhalers. Dry powder inhalers usually contain, in addition to theactive ingredient, a suitable carrier such lactose and, if desired,adjuncts, such as surfactants and/or diluents and/or flow aids and/orlubricants. Metered dose inhalers for dispersing powders usuallycontain, in addition to the active ingredient, a liquid propellant witha boiling point below room temperature and, if desired, adjuncts, suchas liquid or solid non-ionic or anionic surfactants and/or diluents.Pharmaceutical compositions for treatment of the respiratory tract inwhich the pharmacologically active ingredient is in solution (e.g.,either solution for nebulisation or metered dose inhalers) contain, inaddition to this, a suitable propellant, and furthermore, if necessary,an additional solvent and/or a stabiliser. Instead of the propellant,compressed air can also be used, it being possible for this to beproduced as required by means of a suitable compression and expansiondevice.

Parenteral formulations will generally be sterile.

The invention will now be described in greater detail with reference tothe examples and the drawings which are as follows:

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1: ³¹P NMR overlay spectra (202 MHz) of pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoateincubate in deuterated acetone and Trizma buffer and in presence ofCarboxypeptidase Y (Aldrich). The figure shows that the Prodrug israpidly activated by the esterase.

FIG. 2: Half-life calculation for compound pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoatein presence of Carboxypeptidase Y.

FIG. 3: ³¹P NMR (202 MHz) overlay spectra of compound pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate in Human Serum. The figure shows that the prodrug isstable in human serum for at least 665 minutes.

FIG. 4: ³¹P overlay spectra (202 MHz) of pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoatein B95a cell lysate at pH 7.6.

FIG. 5: half-life for pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoatein B95a cell lysate.

FIG. 6: shows the effect of fingolimod prodrugs on Npc1^(−/−) lysosomalexpansion and cholesterol storage phenotypes. Npc1^(−/−) humanfibroblasts were treated for ˜12 hours with 0.2 or 2 μM of eitherfingolimod or prodrugs A, B, C, D, E (see methods for information onprodrug structures) prior to staining with either filipin forcholesterol (A) or lysotracker green for lysosomes (B) and comparisonwith Npc1^(+/+) fibroblasts. Images were quantified to generate graphsshown (C=filipin & D=lysotracker). N=4 for filipin, N=3 for lysotracker.****=p<0.0001, **=p<0.01, *=p<0.05.

DETAILED DESCRIPTION Materials and Methods

General Procedures

All experiments involving water-sensitive compounds were conducted underscrupulously dry conditions. Anhydrous tetrahydrofuran (THF) anddichloromethane were purchased from Aldrich and used directly. Columnchromatography refers to flash column chromatography carried out usingMerck silica gel 60 (40-60 μm) as stationary phase. Proton, carbon, andphosphorus nuclear magnetic resonance (¹H, ¹³C, ³¹P NMR) spectra wererecorded on Bruker Avance spectrometers operating either at 500, 125,and 202 MHz. The solvents used are indicated for each compound. All ¹³Cand ³¹P spectra were recorded proton decoupled. Chemical shifts for ¹Hand ¹³C spectra are in parts per million downfield fromtetramethylsilane. Coupling constants are referred to as J values.Signal splitting patterns are described as singlet (s), doublet (d),triplet (t), quartet (q), broad signal (br), doublet of doublet (dd),doublet of triplet (dt), or multiplet (m). Chemical shifts for 31Pspectra are in parts per million relative to an external phosphoric acidstandard. Some of the proton and carbon NMR signals were splittedbecause of the presence of (phosphate) diastereoisomers in the samples.Electrospray mass spectra were obtained using a Bruker MicroTOF coupledto an Agilent 1100 HPLC system. The electrospray source was operated ata temperature of 130° C. with a desolvation temperature of 300° C., acapillary voltage of 3 kV, and cone voltage of 30 V. Data were collectedin the continuum mode over the mass range 100-2000 amu. Analytical HPLCwas performed on a Thermo Fisher Spectra system 4000 using a RP C-18column Varian Pursuit, 150 mm×4.6 mm, 5.0 μm with detection wavelengthwas 220 nm Mobile phases: Eluent A=H₂O (+0.1% HClO₄), EluentB=Acetonitrile, gradient [time (min.)/% eluent B]: (0/50, 20/85, 22/85,24/00, 25/100, 28/50), flow rate: 0.8 mL/min.

Synthesis of 1-naphthyl Dichlorophosphate

Phosphorus oxychloride (2.59 ml, 27.74 mmol, 1 eq) and 1-naphthol (4 g,27.74 mmol, 1 eq) were stirred in anhydrous Et₂O under an argonatmosphere. Anhydrous Et₃N was added (3.87 ml, 27.74 mmol, 1 eq) at −78°C. After 30 minutes the solution was allowed to warm to roomtemperature. After 3.5 hours after checking the disappear of startingmaterial peak and concomitant formation of the desired products peak by³¹P NMR, the mixture was subjected to vacuum filtration. The solid saltmixture was discarded and Et₂O solvent was removed from the solution invacuo yielding the yellow oil product in 72% yield (5.18 g).C₁₀H₇Cl₂O₆P; M. W: 260.0; ¹H NMR (CDCl₃, 500 MHz): δ8.13-7.37 (7H, m,ArH); ³¹P NMR (CDCl₃, 202 MHz): δ3.73.

Phosphorochloridate Synthesis Standard Procedure

To a stirred solution of the appropriate amino acid ester salt (1equivalent) and the appropriate aryl dichlorophosphate (1 equivalent) inanhydrous CH₂Cl₂ was added dropwise at −78° C. anhydrous Et₃N (2equivalents). Following the addition, the reaction mixture was stirredat −78° C. for 30 min and then at room temperature for 1 h. Formation ofthe desired compound and disappearance of the starting material wasmonitored by ³¹PNMR. After this period the solvent was removed underreduced pressure to give an oil. Most of the aryl phosphorochloridatessynthesised were purified by flash column chromatography on silica gel(eluting with hexane-ethyl acetate 70:30 v/v).

Synthesis of Phenylbenzyloxy-L-leucinyl) Phosphorochioridate

To a stirred solution of L-leucine benzyl ester p-tosylate (3.34 g. 8.5mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq) inanhydrous CH₂Cl₂ (20 ml) was added dropwise at −78° C. anhydrous Et₃N(2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixturewas stirred at −78° C. for 30 min and then at room temperature for 1 h.Formation of the desired compound was monitored by ³¹PNMR. After thisperiod the solvent was removed under reduced pressure to give an oil.The product was then purified by flash column chromatography (elutingwith hexane-ethyl acetate 70:30 v/v) giving the desired compound in 42%yield (1.41 g). C₁₉H₂₃ClNO₄P; M. W: 395.1; ³¹P NMR (CDCl₃, 202 MHz):δ8.45, 8.15.

Synthesis of Phenyl-(benzyloxy-L-prolinyl) Phosphorochioridate

To a stirred solution of L-Proline benzyl ester hydrochloride (2.05 g.8.5 mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq)in anhydrous CH₂Cl₂ (20 ml) was added dropwise at −78° C. anhydrous Et₃N(2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixturewas stirred at −78° C. for 30 min and then at room temperature for 1 h.Formation of the desired compound was monitored by ³¹PNMR. After thisperiod the solvent was removed under reduced pressure to give an oil.The product was then purified by flash column chromatography (elutingwith hexane-ethyl acetate 70:30 v/v) giving the desired compound in 72%yield (2.33 g). C₁₈H₁₉ClNO₄P; M. W: 379.1; ¹H NMR (CDCl₃, 500 MHz):δ7.38-7.16 (10H, m, ArH), 5.16 (2H, m, OCH₂Ph), 4.55 (1H, m, CHNP), 3.51(2H, m, CH₂NP), 2.21 (2H, m, CHCH₂CH₂), 1.96 (2H, m, CHCH₂CH₂); ³¹P NMR(CDCl₃, 202 MHz): δ7.74, 7.67.

Synthesis of Phenyl-(ethoxy-L-alaninyl) Phosphorochioridate

To a stirred solution of L-Alanine ethyl ester hydrochloride (1.31 g.8.5 mmol, 1 eq) and phenyl dichlorophosphate (1.27 ml, 8.5 mmol, 1 eq)in anhydrous CH₂Cl₂ (20 ml) was added dropwise at −78° C. anhydrous Et₃N(2.37 ml, 17 mmol, 2 eq). Following the addition, the reaction mixturewas stirred at −78° C. for 30 min and then at room temperature for 1 h.Formation of the desired compound was monitored by ³¹PNMR. After thisperiod the solvent was removed under reduced pressure to give an oil.The product was then purified by flash column chromatography (elutingwith hexane-ethyl acetate 70:30 v/v) giving the desired compound in 84%yield (2.1 g). C₁₁H₁₅ClNO₄P; M. W: 291.0; ¹H NMR (CDCl₃, 500 MHz):δ7.36-7.21 (5H, m, ArH), 4.38 (1H, m, CHCH₃), 4.22 (2H, m, OCH₂CH₃),1.52 (3H, m, CH₂CH₃), 1.25 (3H, m, CHCH₃); ³¹P NMR (CDCl₃, 202 MHz):δ8.02, 7.70.

Synthesis of 1-Naphthylethoxy-L-alaninyl) Phosphorochioridate

To a stirred solution of L-Alanine ethyl ester hydrochloride (1.31 g.8.5 mmol, 1 eq) and 1-naphthyl dichlorophosphate (1.51 ml, 8.5 mmol, 1eq) in anhydrous CH₂Cl₂ (20 ml) was added dropwise at −78° C. anhydrousEt₃N (2.37 ml, 17 mmol, 2 eq). Following the addition, the reactionmixture was stirred at −78° C. for 30 min and then at room temperaturefor 1 h. Formation of the desired compound was monitored by ³¹PNMR.After this period the solvent was removed under reduced pressure to givean oil. The product was then purified by flash column chromatography(eluting with hexane-ethyl acetate 70:30 v/v) giving the desiredcompound in 64% yield (1.87 g). C₁₅H₁₇ClNO₄P MW: 341.0 ¹H NMR (CDCl₃,500 MHz): δ8.19-7.22 (7H, m, ArH), 4.52 (1H, m, CHCH₃), 4.22 (2H, m,OCH₂CH₃), 1.52 (3H, m, CH₂CH₃), 1.25 (3H, m, CHCH₃); ³¹P NMR (CDCl₃, 202MHz): δ8.28, 8.00.

Phenyl-(methoxy-L-methioninyl) Phosphorochloridate

To a stirred solution of L-methionine methyl ester hydrochloride (3.0 g.14.10 mmol, 1 eq) and phenyl dichlorophosphate (2.11 ml, 14.10 mmol, 1eq) in anhydrous CH₂Cl₂ (100 ml) was added dropwise at −78° C. anhydrousEt₃N (3.94 ml, 28.21 mmol, 2 eq). Following the addition, the reactionmixture was stirred at −78° C. for 30 min and then at room temperaturefor 1 h. Formation of the desired compound was monitored by ³¹PNMR.After this period the solvent was removed under reduced pressure to givean oil. The product was then purified by flash column chromatography(eluting with hexane-ethyl acetate 70:30 v/v) giving the desiredcompound in 80% yield (4.00 g). C₁₃H₁₉ClNO₄P; M. W: 351.79; ¹H NMR(CDCl₃, 500 MHz): δ7.42-7.35 (m, 2H, ArH), 7.33-7.23 (m, 3H, ArH), 4.64(t, J=10.4 Hz, 2H, CHNH), 4.30-4.26 (m, 2H, CH₂CH₃), 2.70-2.54 (m, 2H,CHCH₂), 2.24−2.13 (m, 2H, CH₂S), 2.10 (s, 3H, SCH₃), 1.32 (t, J=7.1 Hz,2H, CH₂CH₃); ³¹P NMR (CDCl₃, 202 MHz): 8.49, 8.36.

Phenyl-(methoxy—O—tert-butyl-L-tyrosinyl) Phosphorochloridate

To a stirred solution of O-tert-butyl L-tyrosine methyl esterhydrochloride (3.0 g. 10.42 mmol, 1 eq) and phenyl dichlorophosphate(1.56 ml, 10.42 mmol, 1 eq) in anhydrous CH₂Cl₂ (100 ml) was addeddropwise at −78° C. anhydrous Et₃N (2.91 ml, 20.85 mmol, 2 eq).Following the addition, the reaction mixture was stirred at −78° C. for30 min and then at room temperature for 1 h. Formation of the desiredcompound was monitored by ³¹PNMR. After this period the solvent wasremoved under reduced pressure to give an oil. The product was thenpurified by flash column chromatography (eluting with hexane-ethylacetate 70:30 v/v) giving the desired compound in 67% yield (2.98 g).C₂₀H₂₅ClNO₅P; M. W: 425.84; ¹H NMR (500 MHz, CDCl₃) δ7.42-7.36 (m, 2H,ArH), 7.25 (m, 3H, ArH), 7.08 (d, J=8.5 Hz, 1H, ArH), 7.04 (d, J=8.5 Hz,1H, ArH), 6.96-6.92 (m, 2H, ArH), 4.50-4.32 (m, 1H, CHNH), 4.24-4.07 (m,1H, CHNH), 3.75 (s, 1H, OMe), 3.73 (s, 1H, OMe), 3.17-3.09 (m, 2H), 1.35(s, 5H), 1.33 (s, 4H); ³¹P NMR (202 MHz, CDCl₃) δ7.90, 7.87.

Phenyl-(dimethoxy-L-glutamyl) Phosphorochloridate

To a stirred solution of L-glutamic acid dimethyl ester hydrochloride(3.0 g. 12.52 mmol, 1 eq) and phenyl dichlorophosphate (1.87 ml, 12.52mmol, 1 eq) in anhydrous CH₂Cl₂ (100 ml) was added dropwise at −78° C.anhydrous Et₃N (3.40 ml, 25.03 mmol, 2 eq). Following the addition, thereaction mixture was stirred at −78° C. for 30 min and then at roomtemperature for 1 h. Formation of the desired compound was monitored by³¹PNMR. After this period the solvent was removed under reduced pressureto give an oil. The product was then purified by flash columnchromatography (eluting with hexane-ethyl acetate 70:30 v/v) giving thedesired compound in 76% yield (3.59 g). C₁₅H₂₁ClNO₆P; M. W: 377.76; ¹HNMR (500 MHz, CDCl₃) δ7.42-7.37 (m, 2H, ArH), 7.30-7.26 (m, 3H, ArH),4.50-4.39 (m, 1H, CHNH), 4.31-4.18 (m, 1H, CHNH), 3.83 (s, 1H, OCH₃),3.81 (s, 1.5H, OCH₃), 3.70 (s, 1.5H, OCH₃), 3.67 (s, 1.5H, OCH₃),2.64-2.35 (m, 2H, CH₂CO₂Me), 2.32-2.22 (m, 1H, CHCH₂), 2.13-2.00 (m, 1H,CHCH₂); ³¹P NMR (202 MHz, CDCl₃) δ8.34, 8.24.

Phenyl-(methoxy-L-tryptophanyl) Phosphorochioridate

FG-33-tryptophan

To a stirred solution of L-Tryptophane methyl ester hydrochloride (2.0g. 7.85 mmol, 1 eq) and phenyl dichlorophosphate (1.17 ml, 7.85 mmol, 1eq) in anhydrous CH₂Cl₂ (100 ml) was added dropwise at −78° C. anhydrousEt₃N (2.13 ml, 15.70 mmol, 2 eq). Following the addition, the reactionmixture was stirred at −78° C. for 30 min and then at room temperaturefor 1 h. Formation of the desired compound was monitored by ³¹PNMR.After this period the solvent was removed under reduced pressure to givean oil. The product was then purified by flash column chromatography(eluting with hexane-ethyl acetate 70:30 v/v) giving the desiredcompound in 72% yield (2.24 g). C₁₈H₁₈ClN₂O₄P; M. W: 392.77; ¹H NMR(CDCl₃, 500 MHz) δ8.11 (s, 1H, NH), 7.51-7.44 (m, 1H, ArH), 7.32-7.20(m, 3H, ArH), 7.19 (s, 1H, ArH), 7.17-6.97 (m, 4H, ArH), 4.51-4.30 (m,1H, CHNH), 4.24-4.05 (m, 1H, CHNH), 3.61 (s, 3H, OCH₃), 3.60 (s, 3H,OCH₃), 3.34-3.21 (m, 2H, CH₂); ³¹P NMR (202 MHz, CDCl₃) δ8.08, 7.99.

Phenyl-(methoxy—O—tert-butyl-L-threoninyl) Phosphorochloridate

To a stirred solution of O-tert-butyl L-Threonine methyl esterhydrochloride (2.0 g. 8.86 mmol, 1 eq) and phenyl dichlorophosphate(1.32 ml, 8.86 mmol, 1 eq) in anhydrous CH₂Cl₂ (100 ml) was addeddropwise at −78° C. anhydrous Et₃N (2.41 ml, 17.72 mmol, 2 eq).Following the addition, the reaction mixture was stirred at −78° C. for30 min and then at room temperature for 1 h. Formation of the desiredcompound was monitored by ³¹PNMR. After this period the solvent wasremoved under reduced pressure to give an oil. The product was thenpurified by flash column chromatography (eluting with hexane-ethylacetate 70:30 v/v) giving the desired compound in 96% yield (3.10 g).C₁₅H₂₃ClNO₅P; M. W: 363.77; ¹H NMR (CDCl₃, 500 MHz) δ7.40-7.35 (m, 2H,Ph), 7.31-7.26 (m, 2H, Ph), 7.26-7.21 (m, 1H, Ph), 4.45 (m, 1H, NH),4.20 (m, 1H, CH₃CH), 3.97 (m, 1H, CHNH), 3.76 (s, 1.5H, OCH₃), 3.74 (s,1.5H, OCH₃), 1.29 (d, J=6.2 Hz, 1.5H, CH₃), 1.27 (d, J=6.2 Hz, 1.5H,CH₃), 1.12 (s, 9H, C(CH₃)₃); ³¹P NMR (CDCl₃, 202 MHz) δ9.71, 8.98.

Phenyl-(methoxy-N-Boc-L-lysinyl) Phosphorochioridate

To a stirred solution of N-Boc-L-lysine methyl ester hydrochloride (2.2g. 7.41 mmol, 1 eq) and phenyl dichlorophosphate (1.11 ml, 7.41 mmol, 1eq) in anhydrous CH₂Cl₂ (100 ml) was added dropwise at −78° C. anhydrousEt₃N (2.01 ml, 14.83 mmol, 2 eq). Following the addition, the reactionmixture was stirred at −78° C. for 30 min and then at room temperaturefor 1 h. Formation of the desired compound was monitored by ³¹PNMR.After this period the solvent was removed under reduced pressure to givean oil. The product was then purified by flash column chromatography(eluting with hexane-ethyl acetate 70:30 v/v) giving the desiredcompound in 89% yield (2.88 g). C₁₈H₂₈ClN₂O₆P; M. W: 674.81; ¹H NMR (500MHz, CDCl₃) δ7.43-7.37 (m, 2H, ArH), 7.31-7.25 (m, 3H, ArH), 4.59 (d,J=27.6 Hz, 1H, CHNH), 4.41-4.27 (m, 1H, CH_(2a)NHBOC), 4.23-4.05 (m, 1H,CH_(2b)NHBOC), 3.82 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.12 (d, J=6.6Hz, 2H, CH₂CHNHP), 1.90 (m, 1H, CH₂), 1.85-1.73 (m, 1H, CH₂), 1.57-1.48(m, 1H, CH₂), 1.48 (s, 9H, C(CH₃)₃); ³¹P NMR (202 MHz, CDCl₃) δ8.40,8.28.

Phosphoramidate Synthesis Using tBuMgCl Standard Procedure

tBuMgCl (1 equivalent) was added dropwise to a solution of primaryalcohol (e.g. fingolimodhydrochloride/1-(4-{[(2S)-2-Amino-3-hydroxy-2-methyl-propoxy]methyl}phenyl)-4-(4-methylphenyl)butan-1-one)(1 equivalent) in anhydrous THF (7 ml) under anhydrous conditions. Themixture was stirred at room temperature for one hour. After this timethe appropriate phosphorochloridate (1 equivalent) in anhydrous THF (2ml) was added dropwise to the stirring reaction mixture. The reactionwas left to stir for 24 hours and then the solvent was removed in vacuoand the desired product was dry-loaded to a column and isolated usingflash chromatography (eluting with methanol-dichloromethane 0:100 v/vincreasing to 10:90 v/v).

EXAMPLE 1

Synthesis of (2S)-methyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)propanoate[Phenyl-(methyloxy-L-alaninyl) Phosphoramidate Fingolimod]

tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution offingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrousTHF (7 ml) under anhydrous conditions. The mixture was stirred at roomtemperature for one hour. After this time Phenyl-(methyloxy-L-alaninyl)phosphorochloridate (403 mg, 1.45 mmol, 1 equivalent) in anhydrous THF(2.6 ml) was added dropwise to the stirring reaction mixture. Thereaction was left to stir for 24 hour and then the solvent was removedin vacuo and the desired product was isolated using flash chromatography(methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving thedesired compound as a mixture of four diastereoisomers in 44% yield(0.35 g). C₂₅H₄₅N₂O₆P; M. W: 548.3; ¹H NMR (CDCl₃, 500 MHz): δ7.29-7.02(9H, m, ArH), 5.19 (1H, m, CHNH), 4.39 (3H, m, OCH₃), 4.18 (1H, m,CHCH₃), 3.81 (2H, m, POCH₂), 3.61 (2H, m, CH₂OH), 2.64 (2H, m,CH₂CH₂Ph), 2.54 (2H, m, CH₂C₇H₁₅), 2.05 (2H, m, CH₂CH₂Ph), 1.61 (2H, m,CH₂C₆H₁₃), 1.42 (1.5H, m, CHCH₃), 1.41 (1.5H, m, CHCH₃), 1.33-1.27 (10H,m, 5× CH₂ C₅H₁₀CH₃), 0.81 (3H, m, CH₃). ³¹P NMR (CDCl₃, 202 MHz): δ3.60,3.47, 3.14, 3.06. MS [ES+] m/z 549.3 [M+H]⁺.

EXAMPLE 2

Synthesis of Benzyl2-(((2-amino-2(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate[Phenyl-(benzyloxy-glycinyl) Phosphoramidate Fingolimod]

tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution offingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrousTHF (7 ml) under anhydrous conditions. The mixture was stirred at roomtemperature for one hour. After this time Phenyl-(benzyloxy-glycinyl)phosphorochloridate (493 mg, 1.45 mmol, 1 equivalent) in anhydrous THF(2.75 ml) was added dropwise to the stirring reaction mixture. Thereaction was left to stir for 24 hours and then the solvent was removedin vacuo and the desired product was isolated using flash chromatography(methanol-dichloromethane 0:100 v/v increasing to 10:90 v/v) giving thedesired compound as a mixture of two diastereoisomers in 17% yield (0.15g). C₃₄H₄₇N₂O₆P; M. W: 610.3; ¹H NMR (CDCl₃, 500 MHz): δ7.32-6.98 (14H,m, ArH), 5.26 (2H, m, OCH₂Ph), 5.19 (1H, m, CHNH), 4.22 (2H, m, CH₂NH),3.81 (2H, m, POCH₂), 3.72 (2H, m, CH₂OH), 2.64 (2H, m, CH₂CH₂Ph), 2.54(2H, m, CH₂C₇H₁₅), 2.05 (2H, m, CH₂CH₂Ph), 1.61 (2H, m, CH₂C₆H₁₃), 1.42(3H, m, CHCH₃), 1.33-1.27 (10H, m, 5× CH₂, C₅H₁₀CH₃), 0.81 (3H, m CH₃).³¹P NMR (CDCl₃, 202 MHz): δ4.27, 3.99; MS [ES+] m/z 611.3 [M+H]⁺.

EXAMPLE 3

Synthesis of (2S)benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate[Phenyl-(benzyloxy-L-leucinyl) Phosphoramidate Fingolimod]

tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution offingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrousTHF (5 ml) under anhydrous conditions. The mixture was stirred at roomtemperature for one hour. After one hour thePhenyl-(benzyloxy-L-leucinyl) phosphorochloridate (574 mg, 1.45 mmol, 1equivalent) in anhydrous THF (3.2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours. After 24hours the solvent was removed in vacuo and the desired product wasisolated using flash chromatography (methanol-dichloromethane 0:100 v/vincreasing to 10:90 v/v) giving the desired compound as a mixture offour diastereoisomers in 23%, yield (0.22 g). C₃₈H₅₅N₂O₆P; M. W: 666.4;¹H NMR (CDCl₃, 500 MHz): δ7.28-6.29 (14H, m, ArH), 4.96 (2H, m, OCH₂Ph),4.39 (2H, m, NH₂), 4.13 (2H, m, POCH₂), 3.91 (1H, m, CHCH₃), 4.02 (1H,m, CHCH₂), 3.42 (2H, m, CH₂OH), 2.52 (2H, m, CH₂CH₂Ph), 2.48 (2H, m,CH₂C₇H₁₅), 1.71 (2H, m, CH₂CH₂Ph), 1.48 (2H, m, CCH₂CH), 1.40 (2H, m,CH₂C₆H₁₃), 1.22-1.17 (10H, m, 5× CH₂, C₅H₁₀CH₃), 0.82 (3H, m, CHCH₃),0.81 (3H, m, CH₃), 0.75 (3H, m, CHCH₃). ³¹P NMR (CDCl₃, 202 MHz): δ4.66,4.46, 4.20, 3.95; MS [ES+] m/z 667.4 [M+H]⁺.

EXAMPLE 4

Synthesis of (2S)benzyl-1-((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)pyrrolidine-2-carboxylate[Phenyl-(benzyloxy-L-prolinyl) Phosphoramidate Fingolimod]

tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution offingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrousTHF (5 ml) under anhydrous conditions. The mixture was stirred at roomtemperature for one hour. After one hour Phenyl-(benzyloxy-L-prolinyl)phosphorochloridate (551 mg, 1.45 mmol, 1 equivalent) in anhydrous THF(4.5 ml) was added dropwise to the stirring reaction mixture. Thereaction was left to stir for 24 hours. After 24 hours the solvent wasremoved in vacuo and the desired product was isolated using flashchromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90v/v) giving the desired compound as a mixture of four diastereoisomersin 20% yield (0.19 g). C₃₇H₅₁N₂O₆P; M. W: 650.4; ¹H NMR (CDCl₃, 500MHz): 7.32-6.98 (14H, m, ArH), 5.16 (2H, m, OCH₂Ph), 4.55 (1H, m, CHN),3.81 (2H, m, POCH₂), 3.61 (2H, m, CH₂OH), 3.51 (2H, m, CH₂NP), 2.64 (2H,m, CH₂CH₂Ph), 2.54 (2H, m, CH₂C₇H₁₃), 2.21 (2H, m, CH₂CH₂Ph), 2.05 (2H,m, CH₂CH₂Ph), 1.96 (2H, m, CHCH₂CH₂), 1.61 (2H, m, CH₂CH₂CH₂), 1.42 (3H,m, CHCH₃), 1.33-1.27 (10H, m, 5× CH₂ C₅H₁₀CH₃); ³¹P NMR (CDCl₃, 202MHz): 2.99, 2.94. MS [ES+] m/z 651.4 [M+H]⁺.

EXAMPLE 5

Synthesis of (2S)ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-propanoate[Phenyl-(ethoxy-L-alaninyl)Phosphoramidate Fingolimod]

tBuMgCl (1.45 ml, 1 equivalent) was added dropwise to a solution offingolimod hydrochloride (500 mg, 1.45 mmol, 1 equivalent) in anhydrousTHF (5 ml) under anhydrous conditions. The mixture was stirred at roomtemperature for one hour. After one hour the Phenyl-(ethoxy-L-alaninyl)phosphorochloridate (423 mg, 1.45 mmol, 1 equivalent) in anhydrous THF(2.8 ml) was added dropwise to the stirring reaction mixture. Thereaction was left to stir for 24 hours. After 24 hours the solvent wasremoved in vacuo and the desired product was isolated using flashchromatography (methanol-dichloromethane 0:100 v/v increasing to 10:90v/v) giving the desired compound as a mixture of four diastereoisomersin 20%, yield (0.16 g). C₃₀H₄₇N₂O₆P; M. W: 562.3; ¹H NMR (CDCl₃, 500MHz): δ7.36-7.05 (9H, m, ArH), 4.22 (3H, m, OCH₂CH₃), 4.05 (2H, m,OCH₂C), 3.65 (1H, m, CHCH₃), 3.45 (2H, m, HOCH₂C), 2.61 (2H, m,CH₂CH₂Ph), 2.58 (2H, m, CH₂CH₂Ph), 1.71 (2H, m, CCH₂CH₂), 1.65 (2H, m,CH₂CH₂CH₂), 1.45 (3H, m, CHCH₃), 1.38 (2H, m, CH₂CH₂CH₂), 1.37 (2H, m,CH₂CH₂CH₂), 1.36 (2H, m, CH₂CH₂CH₂), 1.29 (2H, m, CH₂CH₂CH₂), 1.27 (2H,m, CH₂CH₂CH₂), 1.22 (3H, m, CHCH₃), 0.98 (3H, m, CH₂CH₃). ³¹P NMR(CDCl₃, 202 MHz): δ4.05, 3.84, 3.73, 3.59. MS [ES+] m/z 563.3 [M+H]⁺.

EXAMPLE 6

Synthesis of (2R)-benzyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)Propanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Naphthyl-(benzyloxy-L-Alaninyl) phosphorochloridate (1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in26% yield (0.1 g) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.28, CH₂Cl₂/CH₃OH 95:5 v/v);C₃₉H₅₁N₂O₆P; M. W: 674.81; ¹H NMR (CDCl₃, 500 MHz) δ8.07-7.98 (m, 1H,Naph), 7.80-7.68 (m, 1H, Naph), 7.55 (d, J=8.2 Hz, 1H, Naph) 7.41-7.39(m, 3H, ArH), 7.26-7.17 (m, 6H, ArH), 7.09-6.87 (m, 2H, ArH), 6.83 (m,2H, ArH), 5.30-4.83 (m, 2H, CH₂OBn), 4.23-4.16 (1H, m, CHCH₃) 4.02-3.85(m, 2H, POCH₂), 3.58-3.19 (m, 2H, CH₂OH), 2.53-2.31 (m, 4H, CH₂C₇H₁₅,CH₂CH₂Ph), 1.59-1.41 (m, 4H, CH₂CH₂Ph, CH₂C₆H₁₃), 1.39-1.29 (m, 3H,CHCH₃), 1.28-1.13 (m, 10H, 5× CH₂, C₅H₁₀CH₃), 0.80 (m, 3H, CH₃). ³¹P NMR(CDCl₃, 202 MHz) δ4.59, 4.36, 4.29, 4.12; ¹³C NMR (CDCl₃, 125 MHz)(several signals overlaps) δ173.61, 173.56, 173.45, 173.40, 146.46,140.51, 140.49, 138.64, 138.58, 135.17, 135.15, 134.74, 128.66, 128.59,128.51, 128.44, 128.39, 128.30, 128.18, 128.11, 127.86, 127.81, 125.66,125.52, 125.52, 125.15, 125.11, 125.10, 121.60, 121.46, 115.79, 115.65,77.31, 77.05, 76.80, 68.68, 68.63, 68.48, 67.37, 67.34, 67.32, 65.52,64.74, 64.67, 50.50, 36.86, 35.56, 35.45, 31.95, 31.93, 31.91, 31.61,31.59, 31.58, 29.72, 29.52, 29.50, 29.41, 29.39, 29.37, 29.31, 29.29,29.06, 28.61, 28.56, 28.54, 22.70, 20.82, 14.14; MS [ES+] m/z 675[M+H]⁺, HPLC r.t. 14.1, 14.5, 15.24, 15.80 min.

EXAMPLE 7

Synthesis of (2R)-neopentyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)Propanoate (Prodrug D)

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Naphthyl-(neopentyloxy-L-Alaninyl) phosphorochloridate(1 equivalent) in anhydrous THF (2 ml) was added dropwise to thestirring reaction mixture. The reaction was left to stir for 24 hoursand then the solvent was removed in vacuo and the desired product wasisolated in 29% yield (110 mg) as a mixture of four diastereoisomersusing flash chromatography on a Biotage Isolera, eluting withCH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v). (R_(f)=0.28,CH₂Cl₂/CH₃OH 95:5 v/v). Many signals overlap in ¹H, ¹³C spectra.C₃₇H₅₅N₂O₆P; M. W: 654.81; ¹H NMR (CDCl₃, 500 MHz) δ8.05-8.03 (m, 1H,Naph), 7.77-7.73 (m, 1H, Naph), 7.57 (d, J=8.2 Hz, 1H, Naph), 7.43-4.41(m, 3H, Naph), 7.31-7.29 (m, 1H, Naph), 6.99-6.33 (m, 4H, ArH),4.12-4.04 (m, 1H, CHCH₃), 4.00-3.87 (m, 2H, POCH₂,), 3.83-3.72 (m,1H,CH_(2a)C(CH₃)₃), 3.62-3.67 (m, 1H, CH_(2b)C(CH₃)₃), 3.26-3.17 (2H, m,CH₂OH), 2.49-2.45 (m, 2H, CH₂C₇H₁₅), 2.43-2.36 (m, 2H, CH₂CH₂Ph),1.57-1.48 (m, 2H, CH₂C₆H₁₃), 1.46-1.30 (m, 5H, CH₂CH₂Ph CHCH₃,),1.30-1.14 (m, 10H, C₅H₁₀CH₃), 0.85-0.76 (m, 12H, CH₃, C(CH₃)₃). ³¹P NMR(CDCl₃, 202 MHz) δ4.83, 4.62, 4.58, 4.39; ¹³C NMR (CDCl₃, 125 MHz)δ173.64, 173.59, 146.49, 146.43, 140.46, 138.72, 138.67, 134.76, 128.42,128.40, 128.39, 128.12, 128.08, 127.89, 127.86, 127.82, 125.64, 125.54,125.48, 125.52, 125.14, 125.08, 121.61, 121.51, 121.48, 115.84, 115.71,115.70, 115.55, 74.83, 68.82, 64.98, 50.48, 35.56, 31.92, 31.61, 31.43,29.71, 29.51, 29.39, 29.29, 29.28, 28.51, 26.33, 26.28, 22.69, 22.69,21.21, 21.19, 14.12; MS [ES+] m/z 655.80 [M+H]⁺, HPLC r.t. 22.30, 22.80min.

EXAMPLE 8

Synthesis of methyl3-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate(Prodrug A)

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Naphthyl-(methoxy-L-β-Alaninyl) phosphorochloridate 1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in33% yield (115 mg) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.26, CH₂Cl₂/CH₃OH 95:5 v/v); Manysignals overlap in ¹H, ¹³C spectra; C₃₃H₄₇N₂O₆P, MW: 598.71; ¹H NMR(CDCl₃, 500 MHz) δ8.06-8.02 (m, 1H, Naph), 7.75-7.74 (m, 1H, Naph), 7.57(d, J=8.2 Hz, 1H, Naph), 7.45-7.38 (m, 3H, Naph), 7.31-7.29 (m, 1H,Naph), 7.00-6.91 (m, 4H, ArH), 3.98-3.79 (m, 2H, POCH₂), 3.54 (s, 3H,CH₃), 3.28-3.14 (m, 4H, CH₂OH, CH₂NH), 2.45-2.35 (m, 6H, CH₂CO,CH₂CH₂Ph, CH₂C₇H₁₃), 1.57-1.45(m, 2H, CH₂C₆H₁₃), 1.44-1.36 (m, 2H,CH₂CH₂Ph), 1.29-1.14 (m, 10H, 5× CH₂, C₅H₁₀CH₃), 0.80-0.78 (m, 3H, CH₃).³¹P NMR (202 MHz, Chloroform-d) δ6.45, 6.20; ¹³C NMR (CDCl₃, 125 MHz)δ172.43, 146.55 (d, J_(PH)=7.5 Hz), 146.53 (d, J_(PH)=7.5 Hz), 140.49,140.45, 138.95, 138.90, 134.76, 128.66, 128.42, 128.38, 128.13, 128.10,127.90, 127.88, 125.65, 125.59, 125.54, 125.45, 125.59, 125.54, 125.07,125.00, 121.52, 121.49, 115.67 (d, J_(PC)=2.5 Hz), 115.51, (d,J_(PC)=2.5 Hz), 69.43, 68.94, 65.35, 65.27, 55.88, 51.82, 37.44, 37.41,36.08, 35.96, 35.73, 35.56, 31.92, 31.61, 29.51, 29.40, 29.29, 28.59,28.56, 22.69, 14.13; MS [ES+] m/z 599.71 [M+H]⁺, 621.7 [M+Na]⁺.

EXAMPLE 9

Synthesis of(2R)-neopentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)phosphoryl)amino)propanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time 5,6,7,8-tetrahydro-1-naphthyl-(neopenthyloxy-L-Alaninyl)phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was addeddropwise to the stirring reaction mixture. The reaction was left to stirfor 24 hours and then the solvent was removed in vacuo and the desiredproduct was isolated in 21% yield (100 mg) as a mixture of fourdiastereoisomers using flash chromatography on a Biotage Isolera,eluting with CH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v).(R_(f)=0.42, CH₂Cl₂/CH₃OH 95:5 v/v); Many signals overlap in ¹H, ¹³Cspectra; C₃₇H₅₉N₂O₆P; MW: 658.85; ¹H NMR (CDCl₃, 500 MHz) δ7.78-7.05 (m,7H, ArH), 4.33-3.94 (m, 1H, CHCH₃), 3.94-3.62(m, 4H, POCH₂, CH₂C(CH₃)₃),3.45-3.08 (m, 2H, CH₂OH), 2.64-2.61 (4H, m, 2× CH₂ tetrahydronaph),2.47-2.43 (m, 4H, CH₂CH₂Ph, CH₂C₇H₁₅), 1.73-1.43 (m, 6H, 2× CH₂tetrahydronaph, CH₂C₆H₁₃), 1.38-1.31 (m, 2H, CH₂CH₂Ph) 1.28-1.09 (m,10H, 5× CH₂, C₅H₁₀CH₃), 0.85, 0.84, 0.83, 0.83 (4s, 9H, C(CH₃)₃),0.84-0.78 (m, 3H, CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ4.62, 4.46, 4.23,4.12; ¹³C NMR (CDCl₃, 125 MHz) δ173.79, 173.73, 173.68, 173.66, 173.62,173.60, 148.76, 140.52, 140.51, 139.57, 139.54, 139.50, 138.97, 138.95,138.87, 128.71, 128.67, 128.62, 128.43, 128.13, 128.10, 128.09, 117.13,74.81, 65.36, 65.00, 50.48, 50.37, 36.10, 35.98, 35.56, 31.91, 31.60,31.44, 29.50, 29.47, 29.37, 29.28, 28.61, 28.59, 26.35, 23.55, 23.49,21.26, 21.22, 21.17, 21.12, 14.11; MS [ES+] m/z 659.4 [M+H]⁺, HPLC r.t.23.50, 24.70 min.

EXAMPLE 10

Synthesis ofisopropyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time the Phenyl-(isopropoxy-dimethylglycinyl)phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was addeddropwise to the stirring reaction mixture. The reaction was left to stirfor 24 hours and then the solvent was removed in vacuo and the desiredproduct was isolated in 24% yield (100 mg) as a mixture of fourdiastereoisomers using flash chromatography on a Biotage Isolera,eluting with CH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v).(R_(f)=0.31, CH₂Cl₂/CH₃OH 95:5 v/v); C₃₂H₅₁N₂O₆P; M. W: 590.73; ¹H NMR(CDCl₃, 500 MHz) δ7.26-7.22 (m, 2H, Ph), 7.16-7.13 (m, 2H, Ph),7.10-7.07 (m, 1H, Ph), 7.01-6.95 (m, 2H, ArH), 4.97-4.91 (m, 1H,CH(CH₃)₂), 4.24 (t, J=9.1 Hz, OH), 4.03-3.79 (m, 2H, POCH₂), 3.43-3.17(m, 2H, CH₂OH), 2.60-2.44 (m, 4H, CH₂CH₂Ph, CH₂C₇H₁₅), 1.62-1.42 (m,10H, CH₂C₆H₁₃, CH₂CH₂Ph, C(CH₃)₂), 1.22-1.15 (m, 16H, 5× CH₂ C₅H₁₀CH₃,CH(CH₃)₂), 0.81-0.79 (m, 3H, CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ3.37, 3.15;¹³C NMR (CDCl₃, 125 MHz) δ174.95, 174.86, 174.78, 150.84, 150.78,150.73, 140.51, 138.95, 138.91, 129.71, 129.67, 128.43, 128.16, 128.13,125.10, 125.04, 120.54 (d, J_(PC)=5.0 Hz), 120.43(d, J_(PC)=5.0 Hz),69.58, 69.57, 69.21, 69.16, 68.75, 68.71, 65.26, 65.13, 57.02, 57.01,56.98, 56.97, 56.24, 56.19, 56.15, 56.11, 35.56, 31.90, 31.59, 29.49,29.38, 29.27, 28.62, 27.04, 26.92, 26.87, 26.84, 26.76, 26.74, 26.71,22.67, 21.60, 21.58; MS [ES+] m/z 591.72 [M+H]⁺, HPLC r.t. 17.172 min.

EXAMPLE 11

Synthesis of(2R)-benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)Propanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Naphtyl-(benzyloxy-L-alanilyl) phosphorochloridate (1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in26% yield (128 mg) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.41, CH₂Cl₂/CH₃OH 95:5 v/v);C₃₉H₅₁N₂O₆P; M. Wt: 674.81; ¹H NMR (CDCl₃, 500 MHz) δ8.05-7.99 (m, 1H,Naph), 7.75-7.72 (m, 1H, Naph), 7.56 (d, J=8.3 Hz, 1H, Naph), 7.42-7.47(m, 3H, Naph), 7.30-7.18 (m, 6H, ArH), 6.99-6.94 (m, 2H, ArH), 6.82 (d,J=6.6 Hz, 1H, ArH), 5.08-4.92 (m, 2H, CH₂Ph), 4.10-3.75 (m, 3H, CHCH₃,POCH₂), 3.37-3.07 (m, 2H), 2.47 (td, J=7.5, 3.5 Hz, 3H), 2.51-2.34 (m,4H, CH₂CH₂Ph, CH₂C₇H₁₅), 1.57-1.45 (m, 2H, CH₂C₆H₁₃), 1.42-1.29(m, 5H,CH₂CH₂Ph, CHCH₃), 1.22-1.18 (m, 10H, 5× CH₂, CH₅H₁₀CH₃), 0.80 (m, 1H 3H,CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ4.71, 4.51, 4.47, 4.24; ¹³C NMR (CDCl₃,125 MHz) δ173.42, 173.37, 146.40, 140.49, 140.48, 138.71, 138.64,138.55, 135.64, 135.18, 135.15, 134.74, 134.61, 128.65, 128.59, 128.52,128.50, 128.43, 128.39, 128.37, 128.29, 128.26, 128.18, 128.13, 128.10,128.04, 127.90, 127.85, 127.52, 127.41, 125.64, 125.50, 125.03, 125.92,125.51, 125.13, 125.08, 115.81, 115.78, 115.69, 115.66, 115.55, 115.52,108.51, 67.34, 66.56, 64.69, 50.82, 50.62, 50.49, 50.46, 35.56, 31.92,31.61, 29.52, 29.41, 29.30, 28.53, 22.69, 20.86, 20.82, 20.76, 14.13; MS[ES+] m/z 675.8 [M+H^(]+), HPLC r.t. 17.172 min.

EXAMPLE 12

Synthesis of(2S)-ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-(methylthio)Butanoate (Prodrug E)

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Phenyl-(ethoxy-L-methionyl) phosphorochloridate (1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in37% yield (140 mg) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.36, CH₂Cl₂/CH₃OH 95:5 v/v);C₃₂H₅₁N₂O₆PS; M W: 622.80; ¹H NMR (CDCl₃, 500 MHz) δ 7.25-7.21 (m, 2H,Ph), 7.17-7.13 (m, 2H, Ph), 7.09-7.04 (m, 1H, Ph), 7.10-6.96 (m, 4H,ArH), 4.16-3.78 (m, 5H, POCH₂, OCH₂CH₃, CHNH), 3.50-3.17 (m, 2H, CH₂OH),2.56-2.33 (m, 6H, CH₂CH₂Ph, CH₂C₇H₁₅, CH₂S), 1.98-1.91 (m, 4H, SCH₃,CH_(2a)CH₂S), 1.87-1.69 (m, 1H, CH_(2b)CH₂S), 1.62-1.50 (m, 6H,CH₂CH₂Ph, CH₂C₆H₁₃,), 1.22-1.13(m, 13H, 5× CH₂, C₅H₁₀CH₃, OCH₂CH₃), 0.80(m, 3H, CH₃). ³¹P NMR (CDCl₃, 202 MHz) δ4.56, 4.30, 4.24, 3.98; ¹³C NMR(CDCl₃, 125 MHz) δ 172.88, 172.84, 172.79, 172.78, 172.76, 172.74,150.68, 150.61, 150.56, 140.51, 140.49, 138.99, 138.92, 129.77, 129.73,128.43, 128.17, 128.15, 128.13, 125.25, 125.21, 125.16, 120.50, 120.46,120.40, 120.38, 120.36, 120.34, 120.25, 120.21, 69.51, 69.46, 69.36,69.31, 69.20, 69.16, 65.32, 65.17, 61.80, 61.77, 61.08, 61.03, 58.26,56.00, 55.84, 53.79, 53.72, 53.52, 53.39, 36.11, 36.05, 35.56, 33.53,33.48, 33.43, 31.90, 31.59, 29.75, 29.70, 29.65, 29.49, 29.38, 29.27,28.63, 28.60, 22.67, 15.41, 15.32, 14.25, 14.18, 14.14, 14.11; MS [ES+]m/z 623.3 [M+H]⁺

EXAMPLE 13

Synthesis of(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(4-(tert-butoxy)phenyl)propanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Phenyl-(methoxy—O—tert-butyl-L-tyrosinyl)phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was addeddropwise to the stirring reaction mixture. The reaction was left to stirfor 24 hours and then the solvent was removed in vacuo and the desiredproduct was isolated in 35% yield (150 mg) as a mixture of fourdiastereoisomers using flash chromatography on a Biotage Isolera,eluting with CH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v).(R_(f)=0.32, CH₂Cl₂/CH₃OH 95:5 v/v); C₃₉H₅₇N₂O₇P; MW: 696.85; ¹H NMR(CDCl₃, 500 MHz) δ7.22-7.17 (m, 2H, ArH), 7.06 (d, J=8.4 Hz, 1H, ArH),7.01-6.86 (m, 7H, ArH), 6.82-6.79 (m, 2H, ArH), 4.14-3.98 (m, 1H, CHNH),3.96-3.61 (m, 2H, POCH₂), 3.57, 3.53, 3.51, 3.50 (4s, 4H, OCH₃),3.47-3.24 (m, 2H, CH₂OH), 2.92-2.87 (m, 2H, CHCH₂), 2.64-2.45 (m, 4H,CH₂CH₂Ph, CH₂C₇H₁₅), 1.74-1.64 (m, OH), 1.75-1.49 (m, 4H, CH₂CH₂Ph,CH₂C₆H₁₃), 1.32-1.11 (m, 19H, 5× CH₂, CH₅H₁₀CH₃, C(CH₃)₃), 0.81 (m, 3H,CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ4.11, 3.89, 3.75, 3.11; ¹³C NMR (CDCl₃,125 MHz) δ173.01, 172.98, 172.93, 172.90, 172.56, 172.51, 154.57,150.60, 150.54, 150.47, 140.89, 140.71, 140.64, 140.52, 138.82, 138.61,138.28, 138.18, 130.55, 130.48, 130.40, 129.99, 129.95, 129.90, 129.72,129.70, 129.47, 129.24, 128.64, 128.50, 128.43, 128.16, 128.13, 125.19,125.16, 124.20, 124.16, 123.97, 120.55, 120.51, 120.45, 120.43, 120.41,120.40, 120.35, 120.31, 115.55, 78.44, 75.20, 75.15, 74.95, 74.90,69.04, 69.00, 68.75, 68.71, 68.58, 68.54, 65.37, 64.91, 64.82, 64.79,64.63, 56.14, 55.83, 55.77, 55.43, 52.30, 51.66, 49.93, 49.89, 49.27,39.83, 39.79, 39.77, 39.75, 39.72, 39.70, 36.71, 36.56, 35.72, 35.67,35.57, 31.91, 31.59, 29.71, 29.50, 29.40, 29.28, 28.85, 28.62, 28.58,22.68, 14.12; MS [ES+] m/z 697.3 [M+H]⁺ m/z 697.80 [M+H]⁺

EXAMPLE 14

Synthesis of(2R)-dimethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)pentanedioate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Phenyl-(dimethoxy-L-glutamyl) phosphorochloridate (1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in39% yield (148 mg) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.31 CH₂Cl₂/CH₃OH 95:5 v/v);C₃₂H₄₉N₂O₈P; M Wt: 620.71; ¹H NMR (CDCl₃, 500 MHz) δ7.24-7.19 (m, 2H,Ph), 7.13-7.12 (m, 2H, Ph), 7.01-6.95 (m, 4H, ArH), 4.13-4.03 (m, 1H,CHNH), 4.00-3.83 (m, 3H, CHNH POCH₂), 3.64-3.53 (m, 6H, 2× CO₂CH₃),3.48-3.20 (m, 2H, CH₂OH), 2.54-2.46 (m, 4H, CH₂CH₂Ph, CH₂C₇H₁₅),2.41-2.23 (m, CH₂CH₂CH), 2.14-1.78 (m, 2H, CH₂CH₂CH), 1.63-1.49 (m, 4H,CH₂CH₂Ph, CH₂C₆H₁₃), 1.22-1.18(m, 10H, 5× CH₂, CH₅H₁₀CH₃), 0.81 (m, 3H,CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ4.34, 4.18, 4.08, 3.95; MS [ES+] m/z621.30 [M+H]⁺

EXAMPLE 15

Synthesis of(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(1H-indol-3-yl)propanoate(Prodrug B)

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Phenyl-(methoxy-L-trypthophanyl) phosphorochloridate (1equivalent) in anhydrous THF (2 ml) was added dropwise to the stirringreaction mixture. The reaction was left to stir for 24 hours and thenthe solvent was removed in vacuo and the desired product was isolated in29% yield (110 mg) as a mixture of four diastereoisomers using flashchromatography on a Biotage Isolera, eluting with CH₃OH/CH₂Cl₂ 0:100 v/vincreasing to 10:90 v/v). (R_(f)=0.18, CH₂Cl₂/CH₃OH 95:5 v/v);C₃₇H₅₀N₃O₆P; M. W: 663.78; ¹H NMR (CDCl₃, 500 MHz) δ8.49 (bs, 1H, NH),7.46-7.40 (m, 1H, ArH), 7.24 (m, 2H, ArH), 7.1-6.88 (m, 1H, 10H),4.28-4.16 (m, 1H, CHNH), 3.94-3.64 (m, 3H, CHNH, POCH₂), 3.27-3.01 (m,4H, CH₂OH, CHCH₂), 3.57, 3.56, 3.55, 3.53 (4s, 3H, OCH₃), 2.51-2.38 (m,4H, CH₂CH₂Ph, CH₂C₇H₁₅), 1.52-1.27 (m, 4H, CH₂CH₂Ph, CH₂C₆H₁₃),1.22-1.14 (m, 10H, 2× CH₂, C₅H₁₀CH₃), 0.80 (t, J=6.8 Hz, 3H, CH₃); ³¹PNMR (CDCl₃, 202 MHz) δ4.43, 4.37, 4.19, 4.14; MS [ES+] m/z 664.5 [M+H]⁺.

EXAMPLE 16

Synthesis of(2S,3R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)-3-(tert-butoxy)butanoate

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time Naphthyl-(methoxy—O—tert-butyl-L-threoninyl)phosphorochloridate (1 equivalent) in anhydrous THF (2 ml) was addeddropwise to the stirring reaction mixture. The reaction was left to stirfor 24 hours and then the solvent was removed in vacuo and the desiredproduct was isolated in 4% yield (160 mg) as a mixture of fourdiastereoisomers using flash chromatography on a Biotage Isolera,eluting with CH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v).(R_(f)=0.30, CH₂Cl₂/CH₃OH 95:5 v/v); C₃₄H₅₅N₂O₇P; M W: 634.78; ¹H NMR(CDCl₃, 500 MHz) δ7.35-7.31 (m, 2H, Ph), 7.18-7.154 (m, 2H, Ph),7.01-7.06 (m, 4H, ArH), 4.16-4.12 (m, 1H, CHCH₃), 4.07-3.77 (m, 3H, CHNHPOCH₂), 3.73-3.66 (m, 3H, OCH₃), 3.53-3.141 (m, 2H, CH₂OH), 2.72-2.55(m, 4H, CH₂CH₂Ph, CH₂C₇H₁₅), 1.70-1.55 (m, 4H, CH₂CH₂Ph, CH₂C₆H₁₃),1.31-1.25 (m, 10H, 5× CH₂, CH₅H₁₀CH₃), 1.21-1.16 (m, 3H, CHCH₃),1.11-1.10 (m, 9H, C(CH₃)₃), 0.91-0.88 (m, 3H, CH₃); ³¹P NMR (CDCl₃, 202MHz) δ5.45, 5.18, 4.93, 4.71; MS [ES+] m/z 635.37 [M+H]⁺

EXAMPLE 17

Synthesis of(2R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-6-((tert-butoxycarbonyl)amino)hexanoate (Prodrug C)

tBuMgCl (1 equivalent) was added dropwise to a solution of fingolimodhydrochloride (1 equivalent) in anhydrous THF (7 ml) under anhydrousconditions. The mixture was stirred at room temperature for one hour.After this time the Phenyl-(methoxy-N-Boc-L-lysinyl) phosphorochloridate(1 equivalent) in anhydrous THF (2 ml) was added dropwise to thestirring reaction mixture. The reaction was left to stir for 24 hoursand then the solvent was removed in vacuo and the desired product wasisolated in 39% yield (160 mg) as a mixture of four diastereoisomersusing flash chromatography on a Biotage Isolera, eluting withCH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v). (R_(f)=0.40,CH₂Cl₂/CH₃OH 95:5 v/v); C₃₇H₆₀N₃O₈P; M. W: 705.86; ¹H NMR (CDCl₃, 500MHz) δ7.35-7.32 (m, 2H, Ph), 7.24-7.22 (m, 2H, Ph), 7.20-7.17 (m, 1H,Ph), 7.12-7.06 (m, 4H, ArH), 4.67-4.56 (m, 1H, CHNH), 4.06-4.95 (m, 4H,CH₂NH, POCH₂), 3.71, 3.69, 3.68 (3s, 3H, OCH₃), 3.61-3.42 (m, 2H,CH₂OH), 3.07-3.06 (m, 2H, CH₂CH), 2.64-2.56 (m, 4H, CH₂CH₂Ph, CH₂C₇H₁₅),1.84-1.59 (m, 6H, CH₂CH₂NH, CH₂CH₂Ph, CH₂C₆H₁₃), 1.46 (s, 9H, C(CH₃)₃),1.43-1.39 (m, 2H, CH₂CH₂CH), 1.32-1.24 (m, 10H, C₅H₁₀CH₃), 0.90 (t,J=6.8 Hz, 3H, CH₃); ³¹P NMR (CDCl₃, 202 MHz) δ4.56, 4.32, 4.06, 3.86;¹³C NMR (CDCl₃, 125 MHz) δ173.67, 173.62, 173.45, 156.05, 150.61,140.56, 138.91, 129.77, 129.74, 128.67, 128.52, 128.50, 128.44, 128.16,128.13, 128.10, 128.09, 125.24, 125.20, 125.15, 120.50, 120.46, 120.43,120.39, 120.37, 120.34, 69.24, 69.08, 65.36, 65.30, 58.41, 54.62, 54.40,52.49, 50.79, 40.15, 40.08, 36.05, 33.88, 33.84, 33.78, 31.90, 31.60,29.50, 29.38, 29.28, 28.63, 28.44, 22.68, 22.24, 22.20, 18.44, 14.11; MS[ES+] m/z 706.8 [M+H]⁺

EXAMPLE 18

Synthesis ofS-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)oxy)ethyl)2,2-dimethylpropanethioate (SATE Prodrug)

A. Synthesis of 2,2-Dimethyl-thiopropionic acid S-(2-hydroxy-ethyl)Ester

Pivaloyl chloride (2.5 ml, 20.3 mmol) was added to a stirred solution of2-mercaptoethanol (1.42 ml, 20.3 mmol) and triethylamine (2.83 ml, 20.3mmol) in DCM, cooled at −78° C. The mixture was stirred at −78° C. for 1h. After 1 h the mixture was extracted with water and DCM (3×20 ml). Thecombined organic extracts were dried over MgSO₄and concentrated invacuo. The oily residue was purified by flash column chromatography(eluting with hexane-ethyl acetate 80:20 v/v v/v increasing to 70:30v/v) giving the desired compound (75%, 2.481 g). ¹H NMR (CDCl₃, 500MHz): δ3.98 (2H, m, HOCH₂), 3.03 (2H, m, SCH₂), 2.85 (1H, b, OH), 1.23(9H, s, CCH₃).

B. Synthesis of 2,2-Dimethyl-thiopropionic acidS-[chloro-phenoxy-phosphoryloxy)-ethyl Ester

Dichloro phenyl phosphonate (2.29 ml, 15.31 mmol) was added dropwiseinto a cooled solution at −78° C. of 2,2-Dimethyl-thiopropionic acidS-(2-hydroxy-ethyl) ester (2.481 g, 15.31 mmol) and triethylamine (2.13ml, 15.31 mmol) in THF (20 ml). The reaction was left to warm to roomtemperature and stirred overnight. The white precipitate was filteredoff and the solution was concentrated in vacuo. The crude oil was usedfor the next step without further purification. ³¹P NMR (CDCl₃, 202MHz): δ0.69. ¹H NMR (CDCl₃, 500 MHz): δ7.31 (2H, m, ArH), 7.18 (3H, m,ArH), 4.24 (2H, m, OCH₂), 3.13 (2H, t, SCH₂), 1.17 (9H, s, CCH₃).

C. S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate (SATE Prodrug)

tBuMgCl (1.45 ml, 1.454 mmol) was added dropwise to a solution offingolimod HCl (500 mg, 1.454 mmol) in anhydrous THF (20 ml) underanhydrous conditions. The mixture was stirred at room temperature forone hour. After one hour 2,2-Dimethyl-thiopropionic acidS-[chloro-phenoxy-phosphoryloxy)-ethyl ester (489 mg, 1.454 mmol) inanhydrous THF (5 ml) was added dropwise to the stirring reactionmixture. The reaction was left to stir for 24 hours. After 24 hours thesolvent was removed in vacuo and the desired product was isolated usingflash chromatography (CH₃OH/CH₂Cl₂ 0:100 v/v increasing to 10:90 v/v)giving the desired compound as a mixture of four differentdiastereoisomers in 7% yield (0.076 g). C₃₂H₅₀N₂O₆PS; M. W: 607.31; ¹HNMR (CDCl₃, 500 MHz): δ7.17 (4H, m, ArH), 7.02 (4H, s, ArH), 6.98 (1H,m, ArH), 6.25 (2H, b, CNH₂) 3.95 (2H, q, SCH₂CH₂), 3.72 (4H, m, CCH₂O),3.42 (1H, CH₂OH) 2.95 (2H, t, OCH₂CH₂), 2.55 (4H, m, ArCH₂), 1.89 (2H,m, CCH₂CH₂), 1.59 (2H, quin, CH₂CH₂CH₂), 1.31 (10H, m, CH₂CH₂CH₂), 1.16(9H, s, CCH₃), 0.91 (3H, m, CH₂CH₃). ³¹P NMR (CDCl₃, 202 MHz): δ−5.88.¹³C NMR (CDCl₃, 500 MHz): δ206.41, 152.38, 140.60, 137.89, 129.40,128.43, 128.21, 123.52, 120.07, 64.98, 62.77, 60.81, 46.37, 35.60,33.80, 31.94, 31.63, 29.72, 29.53, 29.48, 29.32, 28.74, 28.67, 28.61,27.63, 27.31, 22.69, 14.13; MS [ES+] m/z 608.8 [M+H]⁺

Other compounds synthesized by methods analogous to those describedabove include:

pentyl(25)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate:

methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate:

Ethyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate:

(3S)-methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-methylpentanoate:

(2S) pentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy) phosphoryl)amino)-3-phenylpropanoate:

EXAMPLE 19

Enzymatic Experiments on pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate

A. Carboxypeptidase Experiment

Blank in acetone d6 alone (No trizma): 1-2 mg of pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate is dissolved in 150 μL of acetone d6 and thesolution transferred into an NMR tube. The ³¹P spectrum is recorded (16scans are sufficient).

The sample in the NMR tube is diluted with 100 μL of Trizma buffer(pH=7.6). 0.1 mg of Carboxypeptidase Y (Aldrich) is then dissolved in150 μL of Trizma buffer and the resulting solution is added to the NMRtube. ³¹P NMR spectra are then recorded (512 scans, 600 sec delay, 20experiments.) FIG. 1

B. Half-Life Determination

The half-life of the compound was also determined in the presence ofCarboxypeptidase Y (see FIG. 2) and was found to be 2109 minutes.

C. Human Serum Stability

Blank in DMSO d6 alone (No trizma).

1-2 mg of pentyl (2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylpheny)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate is dissolved in amixture of 300 μL of DMSO d6 and 100 μL of Trizma buffer (pH=7.6). Thesolution is transferred into an NMR tube. The ³¹P spectrum is recorded(64 scans).

To the blank sample in the NMR tube 150 μL of stock solution of celllysate are added and then ³¹P spectra are recorded (512 scans, 600 secdelay). The results of the above experiment are shown in FIG. 3 andconfirm that pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoateis stable in human serum.

D. B95a Cell Lysate Experiment

4 mg of pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylpheny)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoateare dissolved in a mixture of 150 μL of acetone d6 and 100 μL of trizmabuffer (pH=7.6). The solution is transferred into an NMR tube. The ³¹Pspectrum is recorded (64 scans).

To the blank sample in the NMR tube 150 μL of B95a cell lysate(6.000.000 cell/m L) are added and then ³¹P spectra are recorded at 37Celsius (512 scans, 600 sec delay, 20 experiments).

The overlaid ³¹P spectra are shown in FIG. 4, which makes it clear thatthe compound is processed to release the active monophosphorylatedanalogue. FIG. 5 shows the half-life of the parent compound.

The results of the above experiment confirm that in B95a cell lysatepentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoateis processed with a half-life of 664 minutes.

EXAMPLE 20

Effects of FINGOLIMOD PRODRUGS on Npc1^(−/−) fibroblasts (FIG. 6)

Cell Culture

Glia (mouse astrocytes) were primary cells cultured by Dr E Lloyd-Evansfrom wild-type (Npc1^(+/+)) and NPC1-null (Npc1^(−/−)) mice. Wild-type(NPC1^(+/+)) and NPC1-null (NPC1^(−/−)) human fibroblasts were obtainedfrom the coriell cell bank. All cells were grown as monolayers in ahumidified incubator at 37° C. and 5% CO₂ in complete Dulbecco'sModified Eagle's medium (DMEM). Flask's were used for maintenance andchamber slides (ibidi) and 24-well plates (Greiner) were used for celltreatments.

Cholesterol Staining Using Filipin

Cholesterol was visualized using filipin (filipin complex fromStreptomyces filipinenses), a naturally fluorescent antibiotic thatspecifically binds cholesterol [ref]. PFA-fixed glia were incubated incomplete DMEM with 187.5 μg/ml filipin at room temperature for 30minutes, then washed 3 times in PBS (FIG. 6a ).

Lysotracker Staining for Lysosomes

Human fibroblasts grown in ibidi chamberslides were washed once incomplete Hank's Balanced Salt Solution (HBSS+1 mM HEPES pH7.2, 1 mMCaCl₂, 1 mM MgCl₂) prior to incubation with Lysotracker green(Invitrogen, 200 nM in HBSS), which loads specifically into lysosomes,for 10 minutes at room temperature. Cells were then washed twice incomplete HBSS and imaged live (FIG. 6b ).

The invention claimed is:
 1. A compound of general formula (I) includingall stereoisomers thereof and all isotopic variants thereof:

wherein R¹ is —OAr² or -Q′R^(3′); wherein Ar² is a C₆₋₁₀ aryl or a 5-10membered heteroaryl group optionally substituted with one or moresubstituents selected from OH, halo, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,—O(C₁₋₆ alkyl), —O(C₁₋₆ haloalkyl), NH₂, NH(C₁₋₆ alkyl), N(C₁₋₆ alkyl)₂or SF₅; Q and Q′ are each independently O, S or NR²; R² is H or C₁₋₆alkyl optionally substituted by one or more halo, OH or phenylsubstituents; R³ and R^(3′) are each independently C₁₋₁₀ alkyl or C₁₋₁₀alkyl—C(O)OR¹¹, either of which is optionally substituted by one or moresubstituents R¹²; R¹¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl or benzyl; R¹² is—O—R¹³, —SR¹³, Z, —Z—O—R¹³, —O—Z—R¹³, —Z—R¹³, —C(O)R¹³, —C(O)OR¹³,NR¹³R¹⁴, C(O)NR¹³R¹⁴, —NHC(O)R¹³, —NHC(O)OR¹³, NH(C═NH)NR¹³R¹⁴,—OC(O)—R¹³, —SC(O)R¹³ or —S—S—R¹³; R¹³ and R¹⁴ are each independently Hor C₁₋₆ alkyl; Z is a C₆₋₁₀ aryl or a 5- to 10-membered heteroaryl groupoptionally substituted with one or more substituent selected from haloor OH; or when Q or Q′ is NR², R² and R³ or R² and R^(3′) together withthe nitrogen atom to which they are attached, form a 5- or 6-memberedheterocyclic ring substituted with C(O)OR¹¹, wherein R¹¹ is as definedabove; R⁴ is OH or a group:

where R¹, Q and R³ are as defined above; each of R⁵ and R⁶ isindependently selected from hydrogen or C₁₋₄ alkyl; or R⁵ and R⁶together with the nitrogen atom to which they are attached may form a 5-or 6-membered heterocyclic ring optionally containing a furtherheteroatom selected from N, O or S; Ar¹ is a phenyl or a 5- or6-membered heteroaryl group, either of which is optionally substitutedwith one or more substituents selected from halo, OH, C₁₋₄ alkyl or C₁₋₄haloalkyl; and R⁷ is C₁₋₁₀ alkyl optionally substituted with phenyl or a5- or 6-membered heteroaryl group, wherein the phenyl or heteroarylgroups are optionally substituted with one or more substituents selectedfrom halo, NO₂, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, O(C₁₋₄ alkyl) or phenyloptionally substituted with halo, OH, C₁₋₄ alkyl, C₁₋₄ haloalkyl orO(C₁₋₄ alkyl) and optionally labelled with a fluorescent, visible orisotopic detectable label; or a pharmaceutically or veterinarilyacceptable salt or hydrate thereof.
 2. A compound according to claim 1wherein the asymmetric carbon atom (*) to which the NR⁵R⁶, CH₂R⁴,—CH₂CH₂—Ar¹—R⁷ and the phosphate moiety:

are attached is in the S-orientation.
 3. A compound according to claim 1wherein R¹ is OAr².
 4. A compound according to claim 3 wherein Ar² isphenyl, naphthyl, or tetrahydronaphthyl, any of which is optionallysubstituted with one or more substituents as defined in claim
 1. 5. Acompound according to claim 1 wherein R¹ is -Q′R^(3′).
 6. A compoundaccording to claim 1 wherein Q and/or Q′ (when present) is NR² where R²is H or C₁₋₄ alkyl optionally substituted with one or more halo, OH orphenyl substituents.
 7. A compound according to claim 1 wherein R³and/or R^(3′) (when present) is a group C₁₋₁₀ alkyl—C(O)OR¹¹.
 8. Acompound according to claim 7 wherein R³ and/or R^(3′) (when present) is—C(R^(12a)R^(12b))C(O)OR¹¹ or —C(R^(12a)R^(12b))CH₂C(O)OR¹¹; wherein R¹¹is as defined above; R^(12a) is H or C₁₋₆ alkyl optionally substitutedby a group R¹² as defined above; and R^(12b) is C₁₋₄ alkyl or H.
 9. Acompound according to claim 8 wherein R^(12a) is a side chain of anaturally-occurring amino acid selected from alanine, valine, leucine,isoleucine, methionine, phenylalanine, tyrosine, tryptophan, arginine,histidine, lysine, aspartic acid, glutamic acid, serine, threonine,asparagine, glutamine, cysteine or glycine or a non-natural amino acid.10. A compound according to claim 8 wherein R^(12b) is H, methyl orethyl.
 11. A compound according to claim 9 wherein the R^(12a) sidechain is modified such that OH and/or SH groups are replaced with O—C₁₋₆alkyl or S—C₁₋₆ alkyl and/or carboxylic acid groups are esterified as aC₁₋₆ alkyl or benzyl ester.
 12. A compound according to claim 1 whereinQ is NR², and R² and R³ together with the nitrogen atom to which theyare attached, form a pyrrolidin-1-yl ring substituted at the 2-positionwith C(O)OR¹¹, wherein R¹¹ is as defined in claim 1; and/or wherein R¹is Q′R^(3′), Q′ is NR², and R² and R^(3′) together with the nitrogenatom to which they are attached, form a pyrrolidin-1-yl ring substitutedat the 2-position with C(O)OR¹¹, wherein R¹¹ is as defined in claim 1.13. A compound according to claim 1 wherein Q is O or S and R³ is C₁₋₁₀alkyl substituted with, —OC(O)—R¹³, —SC(O)R¹³ or —S—S—R¹³, where R¹³ isH or C₁₋₆ alkyl; and/or wherein R¹ is Q′R^(3′), Q′ is O or S and R^(3′)is C₁₋₁₀ alkyl substituted with, —OC(O)—R¹³, —SC(O)R¹³ or —S—S—R¹³,where R¹³ is H or C₁₋₆ alkyl.
 14. A compound according to claim 1wherein at least one of R⁵ and R⁶ is H.
 15. A compound according toclaim 1 wherein, independently or in any combination: R⁴ is OH; R⁵ is H;R⁶ is H; R¹ is —OAr² and —Ar² is phenyl; Ar¹ is phenyl and the R⁷ moietyis positioned at the 4-position of the phenyl ring with respect to the—CH₂CH₂—linker group; R⁷ is C₆₋₁₀ alkyl or C₃₋₅ alkyl substituted with(C₁₋₂ alkyl) phenyl; the C* centre has S stereochemistry; and a moiety—O—CH₂—C(CH₂R⁴)(NR⁵R⁶)—CH₂CH₂—Ar¹—R⁷ that is

or an S enantiomer thereof:


16. A compound according to claim 1 selected from the group consistingof: (2S)-methyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)propanoate;benzyl 2-(((2-amino-2(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate; (2S)benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-methylpentanoate; (2S)benzyl-1-((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)pyrrolidine-2-carboxylate; (2S)ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-propanoate; (2R)-benzyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate; (2R)-neopentyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino) propanoate; methyl3-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;(2R)-neopentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)((5,6,7,8-tetrahydronaphthalen-1-yl)oxy)phosphoryl) amino)propanoate;isopropyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-2-methylpropanoate;(2R)-benzyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)propanoate;(2S)-ethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-4-(methylthio) butanoate;(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(4-(tert-butoxy)phenyl)propanoate;(2R)-dimethyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)pentanedioate;(2S)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-(1H-indol-3-yl)propanoate;(2S,3R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(naphthalen-1-yloxy)phosphoryl)amino)-3-(tert-butoxy)butanoate;(2R)-methyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-6-((tert-butoxycarbonyl) amino)hexanoate;S-(2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)oxy)ethyl) 2,2-dimethylpropanethioate; pentyl(2S)-2-[({2-amino-3-hydroxy-2-[2-(4-octylphenyl)ethyl]propoxy}(4-methoxyphenoxy)phosphoryl)amino]propanoate;methyl 2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate; ethyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)acetate; (3S)-methyl2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-methylpentanoate; (2S)pentyl-2-(((2-amino-2-(hydroxymethyl)-4-(4-octylphenyl)butoxy)(phenoxy)phosphoryl)amino)-3-phenylpropanoate; and theirpharmaceutically acceptable salts, hydrates, and stereoisomers.
 17. Aprocess for the preparation of a compound according to claim 1comprising: i. for compounds of general formula (I) in which R⁵ and R⁶are both H: reacting an analogue of the compound of general formula (I)in which one of R⁵ and R⁶ is replaced with —C(O)OR¹⁵, wherein R¹⁵ isC₁₋₆ alkyl or C₆₋₁₄ aryl optionally substituted with one or moresubstituents selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl or halo; to removethe protecting group; or ii. for all compounds of general formula (I):reacting a compound of general formula (II):

wherein R⁵, R⁶, Ar¹ and R⁷ are as defined for general formula (I); witha compound of general formula (III):

wherein: Q, R¹ and R³ are as defined for general formula (I); and X¹ ishalo; wherein the compound of general formula (II) may firstly bereacted with a hindered base, following which the product is reactedwith the compound of general formula (III).
 18. A method for thetreatment of lysosomal storage disorders selected from the groupconsisting of: Niemann-Pick type C1, Niemann-Pick type C2, Niemann-Picktypes A and B, neuronal ceroid lipofuscinoses (NCL), mucolipidoses,lipidoses and sphingolipidoses, Gaucher disease, Fabry disease,Tay-Sachs disease, defective autophagy, accumulation of free cholesteroland endocytic transport defects, mycobacterial diseases, tuberculosisand BCG, the method comprising administering to a patient in need ofsuch treatment an effective amount of a compound according to claim 1.19. A pharmaceutical composition comprising a compound according toclaim 1 and a pharmaceutically acceptable excipient or carrier.
 20. Apharmaceutical composition according to claim 19 which is formulated fororal administration.
 21. The compound according to claim 15 wherein themoiety —O—CH₂—C(CH₂R⁴)(NR⁵R⁶)—CH₂CH₂—Ar¹—R⁷ is the S enantiomer: